Integrated microRNA-mRNA analysis reveals the roles of microRNAs in the muscle fat metabolism of Yanbian cattle.

Fat deposition is an important economic trait in farm animals. However, it is difficult to genetically improve intramuscular fat deposition via trait-based cattle breeding. The main objectives of this study were to analyze the factors about beef flavor, and to detect functional microRNA (miRNA, miR) associated with intramuscular fat deposition in Yanbian cattle. Longissimus dorsi samples from six steers were separated into high- and low-fat groups (n = 3 each) based on the marbling score, and transcriptomic analysis was performed using miRNA sequencing. A total of 33 miRNAs and 38 genes were found to be differentially expressed in the high- and low-fat groups. Quantitative real-time polymerase chain reaction was performed to validate the sequencing results. Integrated miRNA-mRNA analysis revealed that miRNA-associated target genes were primarily associated with skeletal muscle development. However, some of the miRNAs (miR-424 etc.) and genes (ATF3 etc.) were also associated with fat metabolism. A targeted relationship between miR-22-3p and the WFIKKN2 gene and its involvement in adipocyte differentiation were confirmed experimentally. The study findings may provide potential candidate molecular targets for the selection of cattle with improved meat quality.

Bovine bta-microRNA-1271 Promotes Preadipocyte Differentiation by Targeting Activation Transcription Factor 3.

Yanbian yellow cattle are one of the top five largest breeds of cattle in China. We had previously found that bta-miR-1271 is differentially expressed in the longissimus dorsi muscles of Yanbian yellow bulls and steers. However, whether bta-miR-1271 affects bovine fat formation is unclear. In this study, we used target gene prediction, dual-luciferase reporter assay, and transfection-mediated overexpression and inhibition of bta-miR-1271 in a culture of Yanbian yellow cattle preadipocytes to investigate the role of bta-miR-1271 in adipogenesis. We showed that bta-miR-1271 directly targets the 3'-untranslated region (3'-UTR) of the activating transcription factor 3 (ATF3) mRNA and downregulates its expression. Overexpression of bta-miR-1271 enforced by the miRNA mimics promoted triglyceride accumulation and significantly upregulated expression of the adipogenic peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT enhancer-binding protein α (C/EBPα) genes at both the protein and mRNA levels, as demonstrated by RT-qPCR and Western blot analyses. Conversely, inhibition of bta-miR-1271 expression produced the opposite effect. Our results show that bta-miR-1271 regulates differentiation of Yanbian yellow cattle preadipocytes by inhibiting ATF3 expression, which highlights the importance of microRNA-mediated regulation of adipogenesis. miR-1271 and its target gene(s) may provide a new research direction for investigating biological agents affecting intramuscular fat deposition in cattle.

The atypical imaging findings of novel coronavirus pneumonia (COVID-19) and its evolution.

OBJECTIVE: This study aimed to explore the atypical imaging findings of the novel coronavirus pneumonia (COVID-19) and its evolution. MATERIALS AND METHODS: The atypical imaging data of ten patients in our hospital who tested positive for COVID-19 were analyzed retrospectively, and the distribution, morphology, and image evolution of the lesions were analyzed. High-resolution computed tomography (HRCT) was performed in all cases, and the imaging features were analyzed and summarized by two senior radiologists. RESULTS: Of these ten patients, three were male, and seven were female. The age of these patients ranged from 21-53 years, with an average age of 36.3 ± 3.6. The first symptom was fever in nine cases and dry cough in one case. A total of 17 lesions were detected in these ten patients. Five patients had a single lesion, and five patients had multiple lesions, for a total of 12 lesions. Ten lesions (58.82%) were located in the inferior lobe of the right lung, four lesions (23.53%) in the left inferior lobe, two lesions (11.76%) in the left upper lobe, and one lesion (5.88%) in the right middle lobe. Among the five single lesions, two were solid lesions, two were mixed ground-glass lesions, and one was a pure ground-glass lesion. Among the 12 multiple lesions, eight were solid lesions, two were mixed ground-glass lesions, and two were pure ground-glass lesions. Atypical manifestations in image signs: five lesions (29.41%) had single solid and sub-solid nodules, and four lesions (23.53%) had cavitary nodules. Typical manifestation (the presence of "white lung"): three lesions (17.65%) had an air bronchogram, two lesions (11.76%) had crazy-paving signs, two lesions (11.76%) had vascular thickening, and one lesion (5.88%) had halo signs. At reexamination 2-6 days later, 15 lesions (88.24%) had enlarged or increased, and two lesions (11.76%) had decreased or absorbed. CONCLUSIONS: Patients with COVID-19 may have atypical imaging findings. Radiologists should improve their understanding of the novel coronavirus pneumonia to avoid any missed diagnoses.

[Effects of benzyltetrahydropalmatine on ischemia reperfusion with monophasic action potential].

The changes of monophasic action potential (MAP) in ischemic and reperfused feline ventricle in situ were studied. When the feline coronary artery was ligated the MAPD50 was found to be markedly shortened, the T wave and S-T segment were elevated evidently. Within seconds of releasing the coronary occlusion ventricular fibrillation and death occurred in all animals. Intravenous injection of BTHP markedly prolonged the MAPD50 and MAPD90 and reduced the heart rate, while no influence on T wave and S-T segment were observed. After occlusion of the coronary artery, the T wave and S-T segment only increased slightly and the MAPD50 recovered to its control level. During reperfusion the occurrence of ventricular fibrillation was significantly reduced. In contrast, prazosin, verapamil and propranolol did not show significant effect on changes of MAP, but BTHP and Pra, Ver and Pro all showed protective effect on feline ischemic and reperfused myocardia by ligation and reperfusion of the coronary artery with decrease of the number of arrhythmia, ventricular fibrillation and death. The order of antiarrhythmic action of these agents was BTHP = Pra greater than Ver greater than Pro. The total incidence of ventricular fibrillation reduced from the control values of 100% in ischemia/reperfusion heart to 0% (BTHP and Pra), 20% (Ver) and 40% (Pro).

[Effects of linesinine on slow action potentials in myocardium and slow inward current in canine cardiac Purkinje fibers].

Standard microelectrode and two-microelectrodes voltage clamp techniques were used to study the effects of linesinine (Lien), an alkaloid extracted from the green seed embryo of Nelumbo nucifera Gaertn, on slow action potentials (AP) and slow inward current (Isi). Lien 10-100 mumol/L was shown to concentration-dependently decrease the action potential amplitude (APA), the maximal velocity of phase 0 depolarization (Vmax) and prolong the sinus cycle length (SCL) of slow AP in isolated sinoatrial node (SAN) pacemaker cells of rabbits. On both slow APs of rabbit SAN cells and guinea pig papillary muscles partly depolarized by high K+, Bay K 8644 0.03 mumol/L markedly increased the APA and Vmax, which were obviously antagonized by Lien 10 mumol/L. Moreover, Lien 1-100 mumol/L was found to inhibit the Isi of canine cardiac Purkinje fiber in concentration-dependent manner. Lien 3 and 100 mumol/L reduced the peak value of Isi by 14% and 88% respectively. The results suggest that Lien possesses calcium antagonistic effects.

[Effects of amiloride on calcium current in thyroxine-induced hypertrophied rat heart].

AIM: The L-Type calcium currents were investigated in normal, hypertrophied and amiloride-treated rat ventricular myocytes so as to clarify the possible cause of the action potential lengthening that has been reported in thyroxine-induced hypertrophy. The interactions between agents such as thyroxine, Ang II and phenylephrine which induced hypertrophy with amiloride on the calcium current were analyzed. METHODS: Myocardial hypertrophy was induced by i.p. L-thyroxine in rats. The cell length, cell width and cell area were measured by image analysis technique. For recording ICaL, the whole cell patch clamp technique was used. Potassium currents were suppressed by replacing K+ ions with Cs+ ions in intracellular media, and sodium current was blocked by 50 mumol.L-1 tetrodotoxin. RESULTS: The Ca2+ current was found to be larger in hypertrophied cells (783pA) than in normal cells (433pA) and in amiloride-treated cells (429pA). However, no significant difference was observed in current density (6.54, 8.33, 5.74 pA/pF) in normal, hypertrophied and amiloride-treated cells. ICa displayed the same voltage dependence in three cell types. The potential giving 50% of activation (V1/2) moved towards more negative and the slope of activation curve was smaller in hypertrophied cells than in normal cells and amiloride cells. When expressed as percentages, the maximal increases in ICa were obtained with 1 mumol.L-1 Ang II and 100 mumol.L-1 phenylephrine in normal cells (+45.5% and +81.8%). Thyroxine 100 mumol.L-1 showed no effect on ICa. After giving amiloride, Ang II and phenylephrine did not increase the amplitude of ICa. CONCLUSION: At the dose of 0.5 mg.kg-1.d-1, amiloride (p.o.) was shown to prevent the amplitude of ICa from increasing and to be effective against increases of amplitude of ICa by Ang II and phenylephrine.

[The synergistic effect of tetrandrine and rotundine on the action potential of rabbit sinus node dominant pacemaker cell and calcium currents of ventricular myocytes from guinea-pig].

Standard microelectrode technique and the whole-cell clamp technique were used to investigate the effects of tetrandrine and rotundine on the action potential of rabbit sinus node dominant pacemaker cell and calcium currents of ventricular myocytes from guinea-pig. The results indicate that: tetrandrine 1-200 mumol.L-1 and rotundine 3-300 mumol.L-1 decreased the action potential amplitude(APA), the maximal upstroke velocity (Vmax), spontaneous depolarizing velocity of phase 4(SP4) and prolonged the sinus circle length(SCL) in a dose-dependent manner. Tetrandrine 0.1 mumol.L-1 in combination with rotundine 0.3 mumol.L-1 was shown to effectively reduce the ICa of ventricular myotytes from guinea-pig, the rate of inhibition is 19.6%. The results suggest that the two drugs have a good synergistic effect on inhibiting calcium current. These results might also put forward a valuable theoretic base for using the two drugs together in treating patients.

[Protective effects of tetrandrine on ischemic myocardia in anesthetized dogs].

The present study indicates that iv tetrandrine may transiently decrease blood pressure, while other hemodynamic parameters such as LVP, +/- dp/dtmax, LVEDP, HR and CO may not be significantly altered. During coronary occlusion three of five dogs developed ventricular fibrillation (VF) and died in the control group, i.e. incidences of VF and mortality were 60%. While tetrandrine (Tet) appeared to reduce the severity of ischemic injury. It may alleviate arrhythmia and prevent VF and death in four of the five dogs. Tet also attenuated Ca2+ accumulation in myocardial cell, reduced melondialdehyde (MDA) production in ischemic myocardial and decreased CPK release in comparison with the control. It appears that Tet may have significant protective effects against ischemia induced cardiac damage in anesthetized dogs.

[Effects of 4-aminopyridine on calcium currents and sodium currents in guinea pig ventricular myocytes].

AIM: To investigate the effect of 4-aminopyridine (4-AP) on ion channels of myocytes. METHODS: L-type calcium channel and sodium channel currents were recorded in guinea pig single ventricular myocyte using whole-cell patch-clamp techniques. RESULTS: 4-AP, 0.1, 0.5 and 1.0 mmol.L-1 were shown to inhibit L-type calcium channel currents (ICa, L) and sodium channel currents (INa) concentration-dependently. The percentage of inhibition were (11.6 +/- 1.7)%, (37.5 +/- 8.3)% and (54.5 +/- 6.9)% (P < 0.01) respectively for ICa, L, and (22.1 +/- 14.3)% (P < 0.05), (39.4 +/- 8.8)% and (62.3 +/- 6.8)% (P < 0.01) respectively for INa. 4-AP 0.5 mmol.L-1 shifted the I-V curves of ICa, L and INa upwardly. CONCLUSION: 4-AP blocked L-type calcium channel and sodium channels in guinea-pig ventricular myocytes concentration-dependently.

[Blocking effects of benzyltetrahydropalmatine on delayed rectified K+ currents expressed in Xenopus oocytes and in toad oocytes].

The effects of benzyltetrahydropalmatine (BTHP) on delayed rectified K+ currents (Ik) expressed in Xenopus oocytes and Ik of toad (Bufo bufo gargarizans) oocytes were studied. The Ik expressed in Xenopus oocytes was measured after microinjection of mRNA isolated from carp fish (C anratus L.) brains with double -microelectrode voltage clamp technique. The maximum and mean value of Ik expressed in Xenopus oocytes were 600 nA and 360 +/- 104 nA, respectively. BTHP reduced the current amplitude of Ik expressed in Xenopus oocytes in 10-1000 mumol.L-1 dose-dependently, EC50 was 29 mumol.L-1. Also, the reduction of Ik of toad oocytes was 9.1%, 29.1%, 54.7% and 68.6% by BTHP 10, 30, 100 and 1000 mumol.L-1, respectively, EC50 was 33 mumol.L-1. The results showed that BTHP possesses an inhibitory effect on Ik, the main ion mechanism of antiarrhythmic action of BTHP.

[Effects of benzyltetrahydropalmatine on delayed after depolarization and triggered activity and rabbit His-bundle electrogram].

Standard microelectrode techniques were used to study the effects of benzyltetrahydropalmatine (BTHP) on ouabain-induced delayed after depolarization (DAD) and triggered activity in isolated guinea pig papillary muscles. The results indicate that ouabain-induced DAD and triggered activity were abolished by BTHP 100 mumols/L. In anesthetized rabbit ECG heart rates were reduced in a dose-dependent manner from its control value of 288 +/- 14 to 261 +/- 14 (5 mg/kg) and 226 +/- 36 bpm (BTHP 10 mg/kg). P-R interval was prolonged. In His-bundle electrogram, H-V interval and V duration were not affected, but A-H interval was prolonged from 41 +/- 3 to 45 +/- 5 ms.

Use of heterodyne detection to measure optical transmittance over a wide range.

We are developing a heterodyne detection technique to measure optical transmittance with high accuracy over an unprecedented dynamic range. We have measured filters spanning a wide range of transmittances (12 orders of magnitude) and have evaluated the absolute uncertainties and discuss the ultimate accuracies that may be achieved. Our setup uses a two-beam Mach-Zehnder interferometer with acoustooptic frequency shifting to produce a frequency difference between the two light beams. We determine the optical transmittance of a filter by inserting it into one of the interferometer arms and measuring the change in amplitude of the signal at the difference frequency on the interferometer output beam. This method allows direct comparisons between optical and rf attenuators, ultimately tying optical transmittance measurements to rf attenuation standards in an absolute way.

[Effects of benzyltetrahydropalmatine on rat portal vein and guinea pig myocardium].

In isolated rat portal vein, benzyltetrahydrophalmatine (BTHP) 0.3-100 mumol.L-1 dose-dependently increased the spontaneous mechanical activity. However, BTHP 300 mumol.L-1 showed an inhibitory effect on the spontaneous contraction. BTHP 200 mumol.L-1 reversed positive staircase phenomenon to negative ones in left atrium of guinea pig and inhibited the amplitude and Vmax and shortened the action potential duration (APD50) of the slow action potentials induced by high K+ (24 mmol.L-1) in guinea pig papillary muscles. BTHP reduced also the contractile force in papillary muscles of guinea pig. These results suggest that BTHP possesses calcium antagonistic effect in high concentration.

Blocking action of berberine on various receptors in rat anococcygeus muscle.

The contractile responses of rat anococcygeus muscle to phenylephrine, acetylcholine and 5-hydroxytryptamine in relation to berberine and to their specific antagonists were studied separately. The effects of phenylephrine and acetylcholine were blocked by berberine as well as by atropine. But the alpha-blocking action of atropine was exhibited only at high concentration. The anococcygeus muscle response to phenylephrine was competitively inhibited by berberine with a PA2 value of 6.4. The contraction induced by acetylcholine was competitively inhibited by atropine (PA2 = 8.7), whereas berberine, which differs basically from atropine, shifted the dose-response curve for acetylcholine non-parallelly to the right with its maximal response reduced concomitantly. Its pD2(1) value was 4.6. This suggests that the antagonistic action of berberine may be exerted non-specifically through a site beyond the M-cholinergic receptor. Berberine also relaxed the contraction of anococcygeus muscle induced by 5-hydroxytryptamine. The contractile responses to histamine and isoprenaline were seen only at high concentration. In addition, the response to isoprenaline was little affected by propranolol in our experiment.

Effects of benzyltetrahydropalmatine on delayed after depolarization and triggered activity and on His-bundle electrogram and ECG in animal experiment.

Standard microelectrode techniques were used to study the effects of benzyltetrahydropalmatine (BTHP) on ouabain-induced delayed after depolarization (DAD) and triggered activity in isolated guinea papillary muscles. The results indicate that ouabain-induced DAD and triggered activity were abolished by BTHP 100 mumol/L. In anesthetized rabbit ECG heart rate was reduced in a dose-dependent manner from control value of 288 +/- 14 to 261 +/- 14 (BTHP 5 mg/kg) and 226 +/- 36 bpm (BTHP 10 mg/kg). P-R interval was prolonged. In His-bundle electrogram, H-V interval and V duration were not affected, but A-H interval was prolonged from 41 +/- 3 to 45 +/- 5 ms.

A new kalium channel blocker of Chinese medicinal origin--benzyltetrahydropalmatine hydrochloride.

Benzyltetrahydropalmatine hydrochloride (BTHP) exhibited antiarrhythmic action in animal models. I. Electrophysiological effects of BTHP were investigated in various heart preparations. 1) BTHP markedly prolonged functional refractory period and inhibited adrenaline-induced automaticity. It decreased spontaneously beating rate of right atrium and abolished ouabain-induced delayed afterdepolarization and triggered activity of papillary muscle. 2) In standard microelectrode and contractility experiments BTHP concentration-dependently prolonged the action potential duration (APD) and effective refractory period (FRP) between 1-100 mumol/L; the force of contraction remained unchanged. 3) In voltage clamp experiments BTHP 1-100 mumol/L inhibited in dose-dependent manner the Ik with IC50 of 13 mumol/L and inhibited also Is at high concentration. 4) In monophasic action potential (MAP) of feline ventricle MAPD50 and MAPD90 were prolonged by BTHP in normal myocardium, and shortened APD induced by ischemia and reperfusion was restored to normal level. 5) In ECG and His-bundle electrogram heart rate was reduced; P-R and A-H interval were prolonged, but H-V interval and V duration were unaffected. II. BTHP showed a competitive alpha 1-adrenoceptor-blocking effect with pA2 value of 5.8 and 5.86 in rat anococcygeus muscle and rabbit aortic strips respectively. Radioligand binding assays showed that BTHP had affinity for both alpha 1 and alpha 2-adrenoceptor. The results from these experiments show that BTHP is a new K+ channel blocker of Chinese medicinal origin with alpha 1-adrenoceptor-blocking action.