Objectives:Aimed to establish a rapid, high-throughput, automated method for determining the creatine kinase (CK-MM) isoform levels.Methods:Magnetic beads labeled with anti-CK-MM antibodies were combined with alkaline phosphatase-based chemiluminescence detection. Clinical and diagnostic performance validation of the assay was determined by analysis of 998 and 75 dried blood spot samples from healthy newborns and Duchenne muscular dystrophy (DMD) patients, respectively, and the CK activity was also determined. The blank and detection limits, cross-reactivity, recovery rate of the method, intra-and inter-assay coefficient, and the hook effect were evaluated.Results:Blank and detection limits were 17.4 and 39.3 ng/ml, respectively. Cross-reactivity toward CK-MB and CK-BB isoforms was 0.2% and 0.02%, respectively. Intra-and inter-assay coefficients of variation were<1%. Mean recovery was 100.32%, with no hook effect in CK-MM levels<50 000 ng/ml. Overall, the mean CK-MM concentrations in newborns and DMD patients were (27.05±0.97) and (3 720±300.5) ng/ml, respectively. A significant positive correlation between the dried blood spot detected CK-MM levels and total CK enzyme activity, evaluated in corresponding serum samples from the 75 DMD patients, was observed ( r=0.91, P<0.001), ?which is in good agreement with the clinical. Conclusions:An assay for rapid quantitative determination of CK-MM that meets clinical newborn screening requirements was established. It had a good value for application.

Background Flurochloridone (FLC) is toxic to male reproduction and can induce apoptosis of testicular tissue and supporting cells under oxidative stress. Of particular concern is whether nuclear factor-erythrocyte 2-related factor 2/heme oxygenase-1 (Nrf2/HO-1) signaling pathway and nuclear factor kappa B (NFκB) signaling pathway participate this process. Objective To observe apoptosis of testicular tissue and sertoli TM4 cells and alterations of Nrf2/HO-1 and NFκB signaling pathways in mice treated with FLC in vivo/in vitro. Methods (1) Animal experiment. Testis samples were harvested from male C57BL/6 mice after 28-day FLC (0, 3, 15, 75, and 375 mg·kg⁻¹ per day) exposure via oral route. Malondialdehyde (MDA) and superoxide dismutase (SOD) in homogenate of testicular tissue were measured by colorimetry. Apoptosis of testicular tissue was evaluated by TUNEL staining. Expression and distribution of Nrf2 and NFκB were detected by immunohistochemistry. Protein expression levels of Nrf2, HO-1, NAD(P)H: quinone oxidoreductase 1 (NQO1), NFκB, inhibitor of nuclear factor kappa-B kinase subunit beta (IKKβ), and phosphorylated recombinant inhibitory subunit of nuclear factor kappa-B alpha (P-IκBα) in testicular tissue homogenate were determined by Western blotting. (2) Cell experiment. TM4 cell lines were treated with 40, 80, 120, 160, and 200 μmol·L⁻¹ FLC for 6 h, and cell viability was detected by CCK-8. After 6 h exposure to 40, 80, and 160 μmol·L⁻¹ FLC, the apoptosis rate was detected by flow cytometry, and the protein expression levels of Nrf2, HO-1, NQO1, NFκB, IKKβ, and IκBα were detected by Western blotting. Results (1) Animal experiment. Apoptosis occurred in the interstitial and basal parts of spermatogenic tubules in male C57BL/6 mice after 28 days of oral FLC exposure. Compared with the control group, the MDA level in testicular tissue of the 375 mg·kg⁻¹ FLC-treated group was significantly increased (P<0.05), and the SOD activity was significantly decreased (P<0.05). After 375 mg·kg⁻¹ FLC exposure, apoptosis occurred in the interstitial and basal parts of spermatogenic tubules. The results of immunohistochemistry showed the expression of Nrf2 and NFκB in the interstitium and basal part of spermatogenic tubules of the treated groups. Compared with the control group, the protein levels of Nrf2, NQO1, P-IκBα, NFκB, and IKKβ in the 15, 75, and 375 mg·kg^-1 groups were significantly increased (P<0.001), and the HO-1 protein level was significantly increased in the 375 mg·kg⁻¹ group (P<0.001). (2) Cell experiment. Compared with the control group, the TM4 cell viabilities in the 40, 80, 120, 160, and 200 μmol·L⁻¹ FLC-treated groups significantly decreased (P<0.01). The apoptosis rates were significantly increased (P<0.05), and the apoptosis rates increased from 5.7% in the control group to 7.4%, 9.4%, and 11.7% in the 40, 80, and 160 μmol·L⁻¹, respectively. The Nrf2 protein level in the 40 μmol·L⁻¹ group was significantly increased (P<0.01), while the levels significantly decreased in the 80 and 160 μmol·L⁻¹ groups (P<0.01). The HO-1 protein levels in the 40, 80, and 160 μmol·L⁻¹ groups were significantly increased (P<0.01). The level of NQO1 protein in the 40 μmol·L⁻¹ group was significantly increased (P<0.01). The NFκB protein levels were significantly increased in the 80 and 160 μmol·L⁻¹ groups (P<0.001). The IκBα protein levels were significantly decreased in all treated groups (P<0.001). The IKKβ protein had no significant change. Conclusion FLC induces testicular tissue apoptosis, and the process affects Nrf2/HO-1 signaling pathway and NFκB signaling pathway. The in vitro study confirms that FLC could induce apoptosis of TM4 cells and activate Nrf2/HO-1 and NFκB signaling pathways.

Objective:To evaluate the prevention effect of low dose pre-irradiation on irradiation-induced lung injury and its possible mechanism.Methods:Totally 320 6-week-old female C57BL/6j mice were divided into control (0 Gy), low-dose (0.5 Gy), high-dose (20 Gy) and low-dose pre-radiation(0.5 Gy+ 20 Gy)groups by the random number method, with 80 mice in each group. The mice in the low-dose and low-dose pre-irradiation groups were placed in the immobilization device under full consciousness and subjected to 0.5 Gy X-ray whole-body irradiation. 2 weeks later, the 0.5 Gy pre-irradiated mice were anesthetized and subjected to 20 Gy X-rays on chest, as the pre-radiation plus high dose radiation group. The mice in the control group were irradiated with mock irradiation (0 Gy). All mice were terminated at designed time points (24 h, 1 month, 3 months and 5 months) after completion of the irradiation schedule, with 20 mice/group at each time point. Then, lung tissues were taken from mice, and pathological changes were observed by hematoxylin-eosin (HE) staining and Masson′s trichrome staining. RT-qPCR and Western blot were used to detect the expressions of mRNAs and proteins of pulmonary fibrosis-related factors.Results:Pathological changes were observed in the lung tissues 1 month after a single high-dose 20 Gy irradiation, mainly including radiation pneumonitis and a small amount of collagen accumulation, which was more serious than low-dose pre-irradiation group, and these pathological changes became more severe when the time after irradiation increased. Meanwhile, the mRNA and protein levels of proSP-C and HOPX in the low-dose pre-irradiation group were higher than those in the high-dose group, except for proSP-C protein expression at 3 and 5 months post-irradiation. A more significant change was that the mRNA level of TGF-β1 in the high-dose group was 5.8-13.6 times higher than that in the other groups at 5 months after irradiation, as well as β-catenin mRNA ( t=4.22, 5.11, P<0.05). At the same time, in the early period (24 h and 1 month) post-irradiation, the level of vimentin protein in the low-dose pre-irradiation group was significantly higher than that in the high-dose group ( t=6.54, 4.28, P<0.05). Conclusions:When the mice were pre-irradiated with 0.5 Gy X-rays, an adaptive protective response was induced in lung tissues, resulting in the tolerance to subsequent high dose irradiation.

Objective To demonstrate the changes in flexor digitorum and extensor digitorum tension in the affected hands with shear-wave elastography (SWE) before and after manual digitorum sensory stimulation (MDSS) in hemiplegic patients with stroke. Methods A total of 51 hemiplegic post-stroke inpatients in the Department of Rehabilitation Medicine in Second Hospital of Anhui Medical University from April to June, 2020, underwent MDSS completed by a researcher who used a bare thumb and index finger to squeeze each nail bed as well as the sides of each fingertip in the affected hand. The stimulation intensity was the minimum that could cause finger extension without obvious pain, and the interval between two stimulations was five to ten seconds. Muscular tension of the flexor digitorum superficialis, flexor digitorum profundus, flexor pollicis longus and extensor digitorum were assessed with modified Ashworth Scale (MAS) and shear-wave velocity (SWV) of SWE on the affected side before and immediately after MDSS. MAS score was -1 as low muscular tension. Results The MAS scores of all the muscles significantly reduced after MDSS (|Z| > 2.843, P < 0.001); while the changes of SWV were not significantly in all the muscles with initially MAS score of 0 or -1 (t < 1.052, P > 0.05), and it reduced in those muscles with initial MAS scores of one to three (t > 2.672, P < 0.05). The SWV were positively correlated with the MAS scores both before and after MDSS (r > 0.334, P < 0.05). Conclusion MDSS can effectively, immediately, and safely relieves muscle spasms of the flexor digitorum and facilitate active finger extension in the affected hand for hemiplegic patients with stroke. SWE is useful for quantitatively and objectively evaluating muscular tension in the affected hand for hemiplegic patients with stroke.

Objective:To investigate the changes in epidemiological characteristics of common respiratory pathogens in children in Beijing during COVID-19 epidemic.Methods:A total of 9 728 serum samples were collected from cases of acute respiratory infections in Beijing Children′s Hospital from January 2020 to December 2020. Indirect immunofluorescence antibody test was performed to detect IgM antibodies against eight common respiratory pathogens and the test results were statistically analyzed. The eight common respiratory pathogens were influenza virus A (FluA), influenza virus B (FluB), respiratory syncytial virus (RSV), adenovirus (ADV), parainfluenza virus (PIV), Mycoplasma pneumoniae ( Mp), Chlamydia pneumoniae ( Cp) and Legionella pneumophila ( Lp). Results:The detection rate of respiratory pathogens in 9 728 cases was 41.71% (4 058/9 728) and respiratory viruses (FluA, FluB, RSV, ADV and PIV) accounted for 46.18%(2 343/5 074)of all detected pathogens. Mp, FluB and FluA accounted for 84.73%(4 299/5 074)of all detected pathogens, and the detection rates were 24.27% (2 361/9 728), 11.49% (1 118/9 728) and 8.43% (820/9 728), respectively. There were 846 cases positive for two kinds of pathogens, and the most common co-infection was Mp and FluB. The detection rates in male and female were 37.56% (2 089/5 562) and 47.26% (1 969/4 166), respectively. There were significant differences in the total detection rate and the positive rates of PIV and Mp between different sexes ( P<0.05). The detection rate in school-age children (6-12 years old) was the highest (52.26%, 1 535/2 937). The detection rates of respiratory pathogens in different months ranged from 30.12% (203/674) to 49.81% (268/538) with higher rates in autumn and winter [42.45% (1 304/3 072) and 43.29% (1 618/3 738)]. The detection rates of FluA and FluB were higher in summer [11.46% (195/1 701)] and winter [14.63% (547/3738)], respectively. Most of RSV infection occurred in summer [1.35% (23/1 701)], and Mp could be detected all year round, especially in winter and spring [27.21% (1 017/3 738) and 25.64% (312/1 217)]. The detection rate of respiratory pathogens in outpatient group was higher than that in inpatient group [46.48% (1 583/3 406) vs 39.15% (2 475/6 322)]. The detection rate in severe cases was 26.10% (71/272). The detection rates of total pathogens, FluB and Mp were higher in outpatients than in inpatients and the differences were statistically significant ( P<0.05). The detection rates of FluA, PIV and ADV were higher in inpatients than in outpatients and the differences were statistically significant ( P<0.05). The detection rates of total pathogens, FluB and Mp in mild cases were significantly higher than those in severe cases and the differences were statistically significant ( P<0.05). The detection rate of RSV in severe cases was significantly higher than that in mild cases and the difference was statistically significant ( P<0.05). Conclusions:The protective measures taken during the period of regular prevention and control of COVID-19 epidemic could better prevent the spread of respiratory viruses, having a certain impact on the population susceptible to respiratory pathogens and typical seasonal patterns, but had little effect on the prevention and control of Mp. New protective measures needed to be studied to prevent Mp infection in children during epidemical season.

ObjectiveTo investigate the changes of surface electromyography (sEMG) of the flexors and extensors of the affected fingers after manual digitorum sensory stimulation (MDSS) in the hemiplegic patients after stroke. MethodsFrom April to August, 2020, 50 stroke patients in Department of Rehabilitation Medicine, the Second Hospital of Anhui Medical University accepted MDSS on the nail beds and the third knuckles of affected fingers, until the fingers extended actively. The tension of affected flexor pollicis brevis, flexor digitorum superficialis and extensor digitorum were assessed with modified Ashworth Scale (MAS) before and immediately after stimulation; while the root mean square (RMS) of sEMG of bilateral flexor pollicis brevis, flexor digitorum superficialis and extensor digitorum were recorded. ResultsThe MAS score of all the muscles decreased after stimulation (|Z| > 2.699, P < 0.01), while the RMS of affected extensor digitorum increased (t = -2.069, P < 0.05). Compared with the unaffected ones, RMS of affected flexor pollicis brevis and extensor digitorum decreased before and after stimulation (t > 2.450, P < 0.05). ConclusionMDSS may immediately relieve the spasm of flexors of hemiplegic fingers after stroke, which associates with the promoting muscle strength of the extensors.

Objective:To investigate the regulatory effects of endogenous nitric oxide (NO) on the activity of superoxide dismutase-1 (SOD1) and apoptosis of human umbilical vein endothelial cells (HUVECs).Methods:HUVECs were taken as the research object.The endothelial NO synthase (eNOS) short hairpin RNA(shRNA) lentivirus was employed to transfect HUVECs to knock down eNOS.HUVECs were divided in 4 groups: the scramble group, the eNOS shRNA group, the eNOS shRNA + sodium nitroprusside(SNP) group and the eNOS shRNA+ SNP+ tris (2-carboxyethyl) phosphine hydrochloride (TCEP) group.The protein expressions of eNOS and SOD1 dimer/monomer in cells were detected by western blot.The activity of SOD was detected by the enzyme-linked immunosorbent assay.The NO content in cells was detected with NO fluorescence probe.The level of superoxide anion in HUVECs was detected with dihydropyridine (DHE). The terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL) assay was adopted to detect the apoptosis of HUVECs in situ.Results:Compared with the scramble group, the endogenous NO content (2.690±0.420 vs.15.029±2.193, P<0.01), eNOS protein expression (1.000±0.778 vs.3.141±0.199, P<0.01), SOD1 dimer/monomer ratio (4.6±1.0 vs.7.6±2.0, P<0.05) and SOD activity [(0.432±0.254) Carmen′s unit/10 4 cell vs.(1.000±0.116) Carmen′s unit/10 4 cell, P<0.01] were significantly decreased, while the level of intracellular superoxide anion (11.180±1.560 vs.6.146±1.007, P<0.01) and HUVECs apoptosis [75.0 (55.0, 100.0)% vs.0 (0, 0)%, P<0.01] were significantly increased in the eNOS shRNA group.Compared with the eNOS shRNA group, the content of endogenous NO (16.705±0.116 vs.2.690±0.420, P<0.01), the ratio of SOD1 dimer/monomer (7.3±2.0 vs.4.6±1.0, P<0.05) and the activity of SOD [(0.737±0.060) Carmen′s unit/10 4 cell vs.(0.432±0.254) Carmen′s unit/10 4 cell, P<0.05] were significantly increased, while the level of superoxide anion (6.897±1.648 vs.11.180±1.560, P<0.01) and the HUVECs apoptosis [0 (0, 0)% vs.75.0 (55.0, 100.0)%, P<0.01] were significantly decreased in the eNOS shRNA+ SNP group.Compared with the eNOS shRNA + SNP group, the ratio of SOD1 dimer/monomer (4.4±0.9 vs.7.3±2.0, P<0.05) and the activity of SOD [(0.214±0.084) Carmen′s unit/10 4 cell vs.(0.737±0.060) Carmen′s unit/10 4 cell, P<0.01] were significantly decreased, while the level of superoxide anion (10.917±1.552 vs.6.897±1.640, P<0.01) and the apoptosis level of HUVECs[63.6 (55.0, 90.0)% vs.0 (0, 0)%, P<0.01] were significantly increased in the eNOS shRNA+ SNP+ TCEP group.However, there was no significant difference in the NO content (16.112±0.926 vs.16.705±0.116, P>0.05). Conclusions:Endogenous NO could effectively antagonize the apoptosis of endothelial cells by increasing the cysteine-dependent SOD1 dimer/monomer ratio, enhancing SOD activity and inhibiting the accumulation of reactive oxygen species.

Objective:To observe the effect of combining scalp acupuncture with hyperbaric oxygen on serum homocysteine and highly-sensitive c-reactive protein levels and functional recovery after cerebral infarction.Methods:A total of 101 survivors of cerebral infarction were divided randomly into a scalp acupuncture group ( n=33), a hyperbaric oxygen group ( n=34) and a combined treatment group ( n=34). All received routine treatment plus the appropriate supplementary treatment once a day for 10 days. The subjects were evaluated before the experiment as well as 10 and 90 days afterward. The National Institutes of Health′s stroke scale (NIHSS) was used to quantify neurological deficits and the Barthel Index quantified ability in the activities of daily living. Ninety days after the treatment, modified Rankin scale scores were also assigned. The levels of serum homocysteine (Hcy) and highly-sensitive c-reactive protein (hs-CRP) before and after 10 days of treatment were also compared among the 3 groups. Results:The average NIHSS and Barthel Index scores of all three groups had improved significantly after 10 days of treatment and the improvements persisted at the follow-up 3 months later. Both results were significantly better in the combined treatment group than in the scalp acupuncture group at the 90-day follow-up evaluation. The average Rankin score of the combined treatment group was lower at the last follow-up. Compared with before the intervention, the average Hcy of the scalp acupuncture group, the average hs-CRP of the hyperbaric oxygen group, as well as the average Hcy and hs-CRP of the combined treatment group were significantly lower after 10 days of treatment. Compared with the scalp acupuncture group, the average Hcy [(11.68±2.6) μmol/L] of the combined treatment group was significantly lower after the 10 days of treatment.Conclusions:Supplementing scalp acupuncture with hyperbaric oxygen therapy improves the long-term outcomes of cerebral infarction, reducing the level of serum Hcy.

Background: In suckling piglets, transmissible gastroenteritis virus (TGEV) causes lethal diarrhea accompanied by high infection and mortality rates, leading to considerable economic losses. This study explored methods of preventing or inhibiting their production.Bovine antimicrobial peptide-13 (APB-13) has antibacterial, antiviral, and immune functions. Objectives: This study analyzed the efficacy of APB-13 against TGEV through in vivo and in vitro experiments. Methods: The effects of APB-13 toxicity and virus inhibition rate on swine testicular (ST) cells were detected using 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT). The impact of APB-13 on virus replication was examined through the 50% tissue culture infective dose (TCID50 ). The mRNA and protein levels were investigated by real-time quantitative polymerase chain reaction and western blot (WB). Tissue sections were used to detect intestinal morphological development. Results: The safe and effective concentration range of APB-13 on ST cells ranged from 0 to 62.5 µg/mL, and the highest viral inhibitory rate of APB-13 was 74.1%. The log10 TCID50 of 62.5 µg/mL APB-13 was 3.63 lower than that of the virus control. The mRNA and protein expression at 62.5 µg/mL APB-13 was significantly lower than that of the virus control at 24 hpi. Piglets in the APB-13 group showed significantly lower viral shedding than that in the virus control group, and the pathological tissue sections of the jejunum morphology revealed significant differences between the groups. Conclusions APB-13 exhibited good antiviral effects on TGEV invivo and in vitro.

Objective:To evaluate the role of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels in striatum in paclitaxel-induced neuropathic pain and the relationship with GABA B receptors in rats. Methods:Healthy clean-grade male Sprague-Dawley rats, aged 6-9 weeks, weighing 180-220 g, were used in this study.The neuropathic pain model was established by intraperitoneal injection of paclitaxel 2 mg/kg once every 2 days for 7 consecutive days in anesthetized rats, and then intrathecal catheterization was performed.Fifty rats in which intrathecal catheters were successfully implanted were divided into 5 groups ( n=10 each) using a random number table method: paclitaxel group (P group), paclitaxel plus normal saline group(N group), paclitaxel plus lentivirus empty vector group (BV group), paclitaxel plus HCN4 channel lentivirus group (H group), and paclitaxel plus HCN channel inhibitor ZD7288 group (I group). Ten rats of the same age were selected as the blank control group (C group). At 15 days after intraperitoneal injection of paclitaxel, normal saline 20 μl was intrathecally injected in group N, group BV received intrathecal injection of HCN4 channel lentivirus empty vector 1×10 8 TU/ml, 20 μl, group H received intrathecal injection of HCN4 channel lentivirus 1×10 8 TU/ml, 20 μl, and group I received intrathecal injection of ZD728830 μg, 20 μl.The mechanical paw withdrawal threshold (MWT) was measured at 1 day before paclitaxel injection and 14 and 21 days after injection.The cerebral striatum tissues were obtained at T 2, and the expression of HCN4 channel and GABA B receptors was determined by immunohistochemistry and Western blot. Results:Compared with group C, the MWT was significantly decreased, HCN4 channel expression was up-regulated, and GABA B receptor expression was down-regulated in group P ( P<0.05). Compared with group P, the MWT was significantly increased, HCN4 channel expression was down-regulated, and GABA B receptor expression was up-regulated in group H and group I ( P<0.05), and no significant change was found in the parameters mentioned above in group N and group BV ( P>0.05). Conclusion:Up-regulation of expression of HCN4 channels in striatum can induce down-regulation of GABA B receptor expression, which is involved in the pathophysiological mechanism of paclitaxel-induced neuropathic pain in rats.