Osteocalcin activates lipophagy via the ADPN-AMPK/PPARα-mTOR signaling pathway in chicken embryonic hepatocyte.

Fatty liver hemorrhage syndrome (FLHS) is the leading cause of noninfectious mortality in caged layers worldwide. Osteocalcin (OCN) is a protein secreted by osteoblasts, and its undercarboxylated form (ucOCN) acts as a multifunctional hormone that protects laying hens from FLHS. Lipophagy is a form of selective autophagy that breaks down lipid droplets (LDs) through lysosomes, and defective lipophagy is associated with FLHS. The aim of this study was to investigate the effects of ucOCN on the lipophagy of chicken embryonic hepatocytes and associated the function of the adiponectin (ADPN) signaling pathway. In this study, chicken embryonic hepatocytes were divided into 5 groups: control (CONT), fat emulsion (FE, 10% FE, v/v), FE with ucOCN at 1 ng/mL (FE-LOCN), 3 ng/mL (FE-MOCN), and 9 ng/mL (FE-HOCN). In addition, 4 µM AdipoRon, an adiponectin receptor agonist, was used to investigate the function of ADPN. The results showed that compared with CONT group, FE promoted the levels of phosphorylation of mammalian target of rapamycin (p-mTOR) (P < 0.05) and decreased the mRNA expression of ADNP receptors (AdipoR1 and AdipoR2). Compared with FE group, 3 and 9 ng/mL ucOCN inhibited the levels of autophagy adaptor p62 and p-mTOR (P < 0.05), increased the ratios of LC3-II/LC3-I (P < 0.05) and phosphorylated adenosine 5'-monophosphate-activated protein kinase (p-AMPK)/AMPK (P < 0.05), as well as the levels of peroxisome proliferator-activated receptor α (PPAR-α) and ADPN (P < 0.05). In addition, ucOCN at the tested concentrations increased the colocalization of LC3 and LDs in fatty hepatocytes. Administrated 4 µM AdipoRon activated AdipoR1 and AidpoR2 mRNA expression (P < 0.05), decreased the concentrations of triglyceride (P < 0.05), without effects on cell viability (P > 0.05). AdipoRon also increased the LC3-II/LC3-I ratio (P < 0.05) and the levels of p-AMPK/AMPK and PPAR-α (P < 0.05). In conclusion, the results reveal that ucOCN regulates lipid metabolism by activating lipophagy via the ADPN-AMPK/PPARα-mTOR signaling pathway in chicken embryonic hepatocytes. The results may provide new insights for controlling FLHS in laying hens.

[Characteristics of SPECT/CT-derived pulmonary perfusion imaging in chronic pulmonary vascular stenosis with different etiologies].

Objective: To explore the characteristics of pulmonary blood flow perfusion imaging of single photo emission computer tomography/computer tomography (SPECT/CT) in chronic pulmonary vascular Stenosis (CPVS) caused by different etiological factors. Methods: This is a retropective study. Present study screened 50 consecutive cases diagnosed with chronic pulmonary vascular stenosis from January 2019 to January 2020 in the department of cardiology of Gansu Provincial Hospital and underwent SPECT/CT pulmonary blood flow perfusion examination. Thirteen patients were excluded because of pulmonary vascular lesions with a disease course of less than 3 months and poor image quality. According to the etiology, patients were divided into fibrosing mediastinitis (FM) group, Takyasu's arteritis (PTA) group, and chronic thromboembolic pulmonary hypertension/chronic thromboembolic pulmonary disease (CTEPH/CTED) group. The severity of pulmonary blood flow perfusion was evaluated in accordance with the Begic scoring principle in the three groups. The overall Begic score, lung lobe scores among three groups were compared. CT signs of lung SPECT/CT, such as enlargement of hilar lymph node, atelectasis, bronchial stenosis, were also analyzed in three groups. Results: A total of 37 patients with chronic pulmonary vascular stenosis were finally enrolled (18 in the FM group, 5 in the PTA group, and 14 in the CTEPH/CTED group). The total Begic score of pulmonary perfusions was similar among the three groups (F=0.657,P>0.05). There was a statistically significant difference in the left upper lobe Begic score among the three groups (H=4.081, P<0.05). The left upper lobe Begic score was higher in the FM group than in the PTA group (3.44±2.50 vs. 1.60±0.55, P<0.05). As compared to other two groups, patients in FM group were featured with CT signs of higher percent of hilar enlargement (FM group vs. PTA group: 16/18 vs. 1/5, P=0.008; FM group vs. CTEPH/CTED group: 16/18 vs. 3/14, P=0.000 2), enlargement of the pulmonary hilum lymph nodes (FM group vs. PTA group: 14/18 vs. 1/5, P=0.033; FM group vs. CTEPH/CTED group: 14/18 vs. 2/14, P=0.001), and calcification of mediastinal soft tissue (FM group vs. PTA group: 11/18 to 0/5, P=0.037; FM group vs. CTEPH/CTED group: 11/18 vs. 1/14, P=0.003). The proportion of CT signs of bronchial stenosis (9/18 vs. 0/14, P=0.002) and atelectasis (9/18 vs. 1/14, P=0.002) was also higher in the FM group than in the CTEPH/CTED group. In case of abnormal pulmonary blood flow perfusion, the diagnostic accuracy of CT signs hilar enlargement, hilar lymph node enlargement, mediastinal soft tissue calcification, bronchial stenosis, and atelectasis for the diagnosis of FM were 81.1%, 83.8%, 78.4%, 75.7%, and 73.0%, respectively. Conclusion: There is no significant difference in the Begic score of SPECT/CT pulmonary blood flow perfusion imagines among the three groups of patients. Impaired pulmonary blood flow perfusion combined with typical CT signs is useful for identifying patients with FM.

[Short-term efficacy and perioperative safety of catheter-based intervention for pulmonary vein stenosis caused by fibrosing mediastinitis].

Objective: To evaluate the short-term efficacy and perioperative safety of catheter-based intervention in patients with pulmonary vein stenosis caused by fibrosing mediastinitis (FM). Methods: It was a case series study. Consecutive patients with pulmonary vein stenosis caused by FM, who underwent percutaneous pulmonary vein angioplasty in Gansu Provincial Hospital from January 2018 to June 2020, were retrospective enrolled. The baseline characteristics, comorbidities, exercise capacity and hemodynamic data before and after treatment were compared, and the procedural related complications were evaluated. Results: A total of 30 patients ((64.3±7.1) years, 15 males) were included. Sixty-three pulmonary vein stenosis were treated by 32 percutaneous pulmonary vein angioplasty procedures. Forty-four stents were implanted in 41 pulmonary veins after balloon angioplasty, and the diameter of implanted stents was (8.3±1.2)mm. Balloon angioplasty was performed on 22 pulmonary vein stenosis, the mean balloon diameter was (4.2±2.1)mm. The pulmonary vein diameter increased from (2.6±1.3) to (6.6±2.6) mm (P<0.001) and the pressure gradient across the pulmonary vein stenotic segment reduced from 19 (12, 29) to 2 (0, 4) mmHg (1 mmHg=0.133 kPa) (P<0.001) immediately post procedure. The pulmonary vein flow grade was significantly improved compared with baseline (P<0.001). The most common operation related complications were lung injury (44.0% (11/25)) and hemoptysis (18.8% (6/32)), which did not need special treatment. During the 2.0 (1.3, 3.2) months follow-up, the WHO functional class was significantly improved (P<0.05), the 6-minute walking distance increased from (254.8±114.5) m to (342.8±72.4)m (P<0.05), the mean pulmonary arterial pressure decreased from (40.9±8.3) mmHg to (35.4±7.7) mmHg (P<0.01), 17 out of 19 patients with refractory pleural effusion experienced total remission during the follow-up period (P<0.001). CT pulmonary venography was repeated in 17 patients. The incidence of in-stent restenosis of pulmonary vein was 24.0% (6/25). Conclusions: Percutaneous pulmonary vein angioplasty is effective for the treatment of pulmonary vein stenosis caused by fibrosing mediastinitis. However, it's not so safe, procedural related complication should be paid attention to and the rate of in-stent restenosis is relative high during the short-term follow-up.

[Optimizing experimental conditions of graphite furnace atomic absorption spectrometer using orthogonal design for determination of cobalt in urine].

Objective: To select and optimize the conditions of urine cobalt determination by graphite furnace atomic absorption spectroscopy and establish a method for urine cobalt determination. Methods: In April 2020, the matrix modifier and spectrum wavelength were selected by mathematical statistics method, the heating procedure of graphite furnace was optimized, the variance of orthogonal test results was analyzed, and the working curve was quantified by external standard method. Results: The results showed that the diammonium hydrogen phosphate was a matrix modifier with a wavelength of 240.7 nm, and the optimized graphite furnace heating procedure showed a good linear relationship at 5-80 µg/L. The correlation coefficient was 0.9991. The detection limit was 0.9 µg/L, the lowest detection concentration was 1.8 µg/L. The recovery was 94.0%-101.4%, and the precision was 2.6%-5.8%. Conclusion: The orthogonal design method can efficiently and scientifically optimize the optimal combination of the various factor levels of graphite furnace atomic absorption spectrometry. The determination method established based on this has high precision and good accuracy, and can be applied to the determination of cobalt in urine.

[Determination of p-Anisidine in workplace air by HPLC].

Objective: To establish an HPLC method for determining p-Anisidine in workplace air. Methods: An C(18) capillary column was used with the detection wavelength of 240 nm, the methanol solution (v/v=62:38) as mobile phase, the flow rate was 0.9 ml/min, and the injection volume was 20 µl. Results: The linear range (µg/ml) for p-Anisidine was 0~100; correlation coefficient was 0.999 7; the detection limit was 0.15 µg/ml; the average precision of RSD was 4.4%. The sampling efficiency was more than 95% using silicone tube as sampling filter. Sample can be stored at room temperature for more than 7 d. Conclusion: the method appered to be accurate, simple, sensitive and reliable, also have a high sampling efficiency, and can be usd to determine p-anisidine in workplace air simultaneously.

[Correlation of occupational stress with serum levels of immunoglobulins and complement in police].

OBJECTIVE: To investigate the correlation of occupational stress with the serum levels of immunoglobulins(IgG, IgM, and IgA) and complement(C3 and C4). METHODS: In May 2011, convenience sampling and cluster sampling were used to select 225 policemen from a local police station as study subjects. A questionnaire was used to investigate demographic features and occupational stress, and the immunoturbidimetric assay was applied to measure the serum levels of IgG, IgM, IgA, and complement C3 and C4. RESULTS: Positive affectivity was positively correlated with the concentration of IgG(r=0.084, P<0.01); positive affectivity, coping strategy, and sleep quality were positively correlated with the concentration of IgM(r= 0.146, 0.155, and 0.203, all P<0.05); negative affectivity was negatively correlated with the concentration of IgM(r=-0.185, P<0.05). Reward and sleep quality were positively correlated with the concentration of C3(r= 0.172 and 0.285, both P<0.05), and negative affectivity was negatively correlated with the concentration of C3 (r=-0.174, P<0.05); sleep quality was positively correlated with the concentration of C4(r= 0.173, P<0.05), and negative affectivity was negatively correlated with the concentration of C4(r=-0.164, P<0.05). The logistic regression analysis revealed that the risks for a reduced concentration of IgG in the groups with high scores of social support and positive affectivity were 0.460 and 0.495 times those in the groups with low scores, while the risk for a reduced concentration of IgM in the group with a high score of coping strategy was 0.482 times that in the group with a low score. CONCLUSION: Occupational stress is correlated with the serum levels of immunoglobulins and complement.

Structure-function relationship in glycosylated alpha-chymotrypsin as probed by IMAC and IMACE.

Chemical glycosylation of bovine alpha-chymotrypsin, by a glucosamine adduct on the carboxyl group, results in the modification of its catalytic activity. The structural alterations of alpha-chymotrypsin resulting from its glycosylation are studied by immobilized metal-ion affinity chromatography (IMAC) and immobilized metal-ion affinity capillary electrophoresis (IMACE). The chemical glycosylation of alpha-chymotrypsin generates two distinct subpopulations of the protein: one which totally loses the initial affinity for IDA-Cu(II) and another which exhibits an increased affinity for the metal chelate ligand.

Effects of tanshinone II-A sulfonate on adhesion molecule expression of endothelial cells and platelets in vitro.

AIM: To study the action of tanshinone II-A sulfonate (Tan) on adhesion molecule expression by cultured endothelial cells and platelets. METHODS: Tumor necrosis factor alpha (TNF-alpha)-induced ICAM-1 expression on the cell surface and endothelial adhesivity toward HL-60 cells were studied using human umbilical vein endothelial cells (HUVEC). Thrombin-induced expression of platelet P-selectin was studied using human blood platelets. Adhesion molecule expression on the cell surface was measured by flow cytometry. The number of HL-60 cells adhering to the HUVEC monolayer was determined by liquid scintillation spectroscopy. RESULTS: Pretreatment of HUVEC with TNF-alpha significantly enhanced ICAM-1 expression and increased HL-60 cells adhesion to HUVEC from 4.6% +/- 0.7% to 30% +/- 6%. Tan (25-200 mumol.L-1) inhibited the effects of TNF-alpha in a concentration-dependent manner. Tan also inhibited the increase of P-selectin expression of thrombin-activated platelets in a concentration-dependent manner. CONCLUSION: Tan inhibited expression of adhesion molecules (ICAM-1, P-selectin) in HUVEC and in human blood platelets.

Esters and amides of 6-(chloromethyl)-2-oxo-2H-1-benzopyran-3-carboxylic acid as inhibitors of alpha-chymotrypsin: significance of the "aromatic" nature of the novel ester-type coumarin for strong inhibitory activity.

A series of esters and amides of 6-(chloromethyl)-2-oxo-2H-1-benzopyran-3-carboxylic acid were synthesized and evaluated in vitro for their inhibitory activity toward bovine alpha-chymotrypsin and human leukocyte elastase. Both series behaved as time-dependent inhibitors of alpha-chymotrypsin, but ester-type coumarins were clearly more efficient than the corresponding amides in inactivating the serine proteinase. The best inactivations were observed with "aromatic" esters, in particular with meta-substituted phenyl esters such as m-chlorophenyl 6-(chloromethyl)-2-oxo-2H-1-benzopyran-3-carboxylate, which appears to be one of the most powerful inactivators of alpha-chymotrypsin yet reported (kinact/KI = 760,000 M-1 S-1 at pH 7.5 and 25 degrees C). Usually, the coumarin derivatives failed to inhibit significantly human leukocyte elastase. As a result, the reported series of aromatic coumarinic esters behaves as a new chemical family of selective alpha-chymotrypsin inhibitors.

Effects of Panax notoginseng saponins on posthypoxic cell damage of neurons in vitro.

AIM: To study cerebral protective mechanism of Panax notoginseng saponins (PNS). METHODS: Cultured neurons of chick embryo cerebral hemisphere were used as an in vitro system for investigating the effects of PNS. The hypoxic cell damage of neurons cultured were induced by NaCN. The levels of adenosine triphosphate (ATP) were determined with HPLC. PNS was added 30 min before, beginning or after hypoxia. RESULTS: PNS 50 and 100 mg L-1 retarded the break down of ATP of cultured neurons after 2-h hypoxia for 11.3 +/- 1.5 (P < 0.05) and 12.8 +/- 2.2 mumol/g protein (P < 0.01), respectively and accelerated the restoration of ATP during 30-min reoxygenation for 21.0 +/- 2.0 (P < 0.05) and 22.7 +/- 2.6 mumol/g protein (P < 0.01), respectively. PNS also reduced the release of creatine kinase (CK) from 75 +/- 8 kU L-1/g protein to 52 +/- 6 (P < 0.05) and 41 +/- 3 kU L-1/mg protein (P < 0.01), respectively and promoted the restoration of ATP of neurons 20 h after hypoxia when administered in the beginning of hypoxia from 13.0 +/- 0.9 mumol/g protein to 18.1 +/- 1.4 and 20.5 +/- 2.1 mumol/g protein (P < 0.01), respectively. PNS still promoted the restoration of ATP from 13.0 +/- 0.9 nmol/mg protein to 14.9 +/- 1.0 and 18.3 +/- 0.7 nmol/mg protein (P < 0.01), respectively and reduced (PNS 100 mg L-1) the CK release of neurons 20 h after hypoxia even when added in the recovery. CONCLUSION: The protection against hypoxic damage of PNS was related to improving energy metabolism, preserving the structural integrity of neurons.

Dynamic balance changes between elastase and antiprotease in the early stages after smoke inhalation injury.

Following the use of a rabbit smoke inhalation injury model established in this institute, there were marked reductions in elastase activities in neutrophils and alveolar macrophages; rapid increases in elastase activity in broncho-alveolar lavage fluid (BALF); reductions of serum trypsin inhibitory capacity; a decrease of Pao2 and an increase of PaCO2, and marked increases of lung water volume. Significant correlations were found between the increased extravascular lung water content and the rising elastase activity in BALF. It seems probable that the imbalance between elastase and antiprotease played an important role in the development of acute lung injury after smoke inhalation.

[Experimental study on combined treatment in smoke inhalation injury].

Forty-two healthy dogs were randomly divided equally into a control group (CG) and a treated group (TG). All were inflicted with severe smoke inhalation injury and pulmonary was demonstrated. The dogs in TG were treated with injection of shenmai zhusheye, ketoprofen, anisodamine, sodium aesculin, hydrocortisone succinate, vitamin C and E, penicillin, amikacin, oxygen inhalation and airway suctioning after injury. The results showed that the increase in extravascular lung water volume, lung edema shadow in x-ray films, elevation of lung vascular resistance, carbonemia, hypoxemia, respiratory alkalosis, metabolic acidosis, dyspnea, dry and moist rales of the lungs, reduction of tidal volume, etc, were markedly improved in TG as compared with that of CG. It indicated that the pulmonary edema and lung dysfunction was markedly ameliorated in TG. The mortality was 19.1% in TG, which was significant lower than that of CG which was 47.6%.

A full-to-empty mode controller for the pneumatically-driven blood pump.

Presented in this paper is a microprocessor-based controller developed based upon the theory of finite state automata to activate the pneumatically-driven diaphragm blood pump in the full-to-empty mode. Under normal situations, each pumping cycle of the blood pump can be divided into several phases in a proper sequence, and such phase information is contained in the driving air pressure waveform. The controller tries to match the sequential phases with the pressure waveform sampled at real time in an attempt to search for the end-ejection or end-filling of the blood pump. If either the end-ejection or the end-filling can be found, the controller sends triggering signals to the driving unit, and if the sampled pressure waveform can not be matched with the sequential phases or the controller fails to detect the end-ejection or end-filling, the controller activates the driving unit by the parameters of the last measured pumping cycle; thus, the driving unit triggers the blood pump without any stop. The result of laboratory bench tests demonstrates its ability in finding the end-ejection and end-filling points and the reliability of the fail-safe measure.

A generic algorithm for finding restriction sites within DNA sequences.

This paper describes a generic algorithm for finding restriction sites within DNA sequences. The 'genericity' of the algorithm is made possible through the use of set theory. Basic elements of DNA sequences, i.e. nucleotides (bases), are represented in sets, and DNA sequences, whether specific, ambiguous or even protein-coding, are represented as sequences of those sets. The set intersection operation demonstrates its ability to perform pattern-matching correctly on various DNA sequences. The performance analysis showed that the degree of complexity of the pattern matching is reduced from exponential to linear. An example is given to show the actual and potential restriction sites, derived by the generic algorithm, in the DNA sequence template coding for a synthetic calmodulin.

Changes of length of elastic fibers in rabbit lungs after smoke inhalation injury.

Pulmonary morphometry was determined in rabbit lungs after smoke inhalation. Twenty-four hours after injury, changes of the total lung volume (TLV) and the total length of all parenchymal elastic fibers (LT) were measured. Concomitant changes in arterial blood gas levels and pathomorphology of lung tissues were also studied. It was found that alveolar walls were broken and became thinner, and that alveoli were dilated. TLV was markedly increased and LT was markedly decreased as compared to normal lungs (P less than 0.05). These findings suggest that proteinases, especially elastase, released from phagocytes may play an important role in acute lung injury after smoke inhalation.

Blood gas studies in dogs with severe steam inhalation injury.

The dynamic changes in blood gases in 34 dogs suffering severe steam inhalation injury were studied during the first 14 days post-burn to determine the effects of inhalation injury on lung function. Nineteen dogs received only severe steam inhalation injury, while 15 other dogs received both 30 per cent total body surface area (TBSA) deep burns and steam inhalation injury. The results showed marked reductions of PaO2, PaCO2, AB, SBE, T-CO2, SB, BBp, pHNR and SatO2 (P less than 0.05 approximately 0.01), whereas the P(A-a)O2 increased (P less than 0.05 approximately 0.01) within week 1 post-burn in both groups. These data suggest that the injured animals were suffering from hypoxaemia and respiratory alkalosis induced by disturbance of the gas-exchange function of the lungs. There was also metabolic acidosis, which was closely related to high fever, excessive catabolism, and particularly to hypoxaemia in week 1 post-burn. If the animals survived through the critical period (1-7 days post-burn), most of the abnormal gas parameters then gradually returned to the pre-burn levels. It is clear, therefore, that blood gas monitoring in patients with inhalation injury is not only of diagnostic importance but also of prognostic value.