M2 protein of influenza A virus is encoded by a spliced mRNA derived from RNA segment 7 and plays an important role in influenza virus replication. It is also a target molecule of anti-virus drugs. We extracted the viral genome RNAs from MDCK cells infected with swine influenza A virus (SIV) H3N2 subtype and amplified the SIV M2 gene by reverse transcriptase-polymerase chain reaction using the isloated viral genome RNAs as template. The amplified cDNA was cloned into a prokaryotic expression vector pET-28a(+) (designated pET-28a(+)-M2) and a eukaryotic expression vector p3xFLAG-CMV-7.1 (designated p3xFLAG-CMV-7.1-M2), respectively. The resulted constructs were confirmed by restriction enzyme digestion and DNA sequencing analysis. We then transformed the plasmid pET-28a(+)-M2 into Escherichia coli BL21 (DE3) strain and expressed it by adding 1 mmol/L of IPTG (isopropyl-beta-D-thiogalactopyranoside). The recombinant M2 protein was purified from the induced bacterial cells using Ni(2+) affinity chromatography. Wistar rats were immunized with the purified M2 protein for producing polyclonal antibodies specific for it. Western blotting analysis and immunofluorescence analysis showed that the produced antibodies were capable of reacting with M2 protein expressed in p3xFLAG-CMV-7.1-M2-transfected cells as well as that synthesized in SIV-infected cells. We also transfected plasmid p3xFLAG-CMV-7.1-M2 into Vero cells and analyzed its subcellular localization by immunofluorescence. The M2 protein expressed in the Vero cells was 20 kDa in size and dominantly localized in the cytoplasm, showing a similar distribution to that in SIV-infected cells. Western blotting analysis of SIV-infected cells suggested that M2 was a late phase protein, which was detectable 12 h post-infection, later than NS1, NP and M1 proteins. It would be a potential molecular indicator of late phases replication of virus. Our results would be useful for studying the biological function of M2 protein in SIV replication.
Animals ; Antibodies, Monoclonal ; biosynthesis ; Cercopithecus aethiops ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Influenza A Virus, H3N2 Subtype ; genetics ; RNA ; biosynthesis ; genetics ; Rats ; Rats, Wistar ; Recombinant Proteins ; biosynthesis ; genetics ; immunology ; Swine ; Transfection ; Vero Cells ; Viral Matrix Proteins ; biosynthesis ; genetics ; Virus Replication ; genetics

Objective:To investigate the preoperative risk factors affecting early extremity blood supply after repair of major arterial injury so as to provide clues for prevention of limb ischemia.Methods:The clinical data were retrospectively analyzed of the 139 patients (140 extremities) with major extremity arterial injury who had been admitted to Department of Microsurgery, Orthopaedic Trauma and Hand Surgery, The First Hospital Affiliated to Sun Yat-sen University from January 2003 to December 2019. There were 112 males and 27 females, with a mean age of 30 (20, 44) years. The primary outcome was the early status of blood supply to the injured extremity (48 hours after surgery). Univariate analysis was conducted of such factors as gender, age, ischemia time, injury mechanism, injury site, fracture, soft tissue lesion, and duration of surgery. The significant factors ( P<0.1) were then analyzed by logistic regression, and P<0.05 was considered statistically significant. Results:Ischemia happened in 44 (31.4%, 44/140) extremities within 48 hours after surgery. There were significant differences in injury mechanism, ischemia time, fracture, and soft tissue lesion between patients with and without postoperative extremity ischemia ( P<0.05). Logistic regression analysis indicated that blunt injury ( OR=5.639, 95% CI: 1.068 to 29.761, P=0.042) and soft tissue lesion ( OR=12.568, 95% CI: 3.402 to 46.431, P<0.001) were significant preoperative risk factors affecting the early blood supply after repair of major extremity arterial injury. Conclusion:As blunt injury and soft tissue defect are preoperative risk factors for early extremity ischemia after repair of major extremity arterial injury, surgeons should pay more attention to them when assessing patients and making repair protocols.

Depressive disorder ranks as a major bur-den of disease worldwide,yet the current antidepressant medications are limited by frequent non-responsiveness and significant side effects.The lateral septum(LS)is thought to control of depression,however,the cellular and circuit substrates are largely unknown.Here,we identified a subpopulation of LS GABAergic adenosine A2A receptors(A2AR)-positive neurons mediating depres-sive symptoms via direct projects to the lateral habenula(LHb)and the dorsomedial hypothalamus(DMH).Activa-tion of A2AR in the LS augmented the spiking frequency of A2AR-positive neurons leading to a decreased activation of surrounding neurons and the bi-directional manipula-tion of LS-A2AR activity demonstrated that LS-A2ARs are necessary and sufficient to trigger depressive pheno-types.Thus,the optogenetic modulation(stimulation or inhibition)of LS-A2AR-positive neuronal activity or LS-A2AR-positive neurons projection terminals to the LHb or DMH,phenocopied depressive behaviors.Moreover,A2AR are upregulated in the LS in two male mouse mod-els of repeated stress-induced depression.This identifica-tion that aberrantly increased A2AR signaling in the LS is a critical upstream regulator of repeated stress-induced depressive-like behaviors provides a neurophysiological and circuit-based justification of the antidepressant poten-tial of A2AR antagonists,prompting their clinical transla-tion.

ObjectiveTo analyze the clinical application hotspots， development trends， compatibility characteristics， application rules， and formulation mechanisms of the Chinese marine drug pair Haliotidis Concha-Oystreae Concha in order to provide references for its clinical medication and further research. MethodBy means of various modern literature databases such as China National Knowledge Infrastructure （CNKI）， modern clinical prescriptions containing Haliotidis Concha-Oystreae Concha， as well as the clinical application hotspots， were retrieved， followed by visualized analysis of hotspots and development trends of their clinical applications using Citespace. The drug composition， efficacy and indications， and drug dosages in the prescriptions were statistically analyzed. Additionally， various statistical software including SPSS Modeler 18.0 were employed to analyze the indications， syndromes， and formulation rules of Haliotidis Concha-Oystreae Concha. ResultThe visualized analysis included 90 articles， revealing a gradual decrease in publications in this field in recent years. Key clinical application keywords were identified as hypertension， collateral deficiency producing wind， insomnia， etc. Eighty clinical prescriptions were retrieved， involving 121 drugs. Frequency analysis of compatibility demonstrated that the top 10 drugs were Uncariae Ramulus cum Uncis， Gastrodiae Rhizoma， Os Draconis， Achyranthis Bidentatae Radix， Paeoniae Radix Alba， Chrysanthemi Flos， Scutellariae Radix， Gardeniae Frucuts， Glycyrrhizae Radix et Rhizoma， and Polygoni Multiflori Caulis. Association rule analysis showed that core combinations included "Uncariae Ramulus cum Uncis-Achyranthis Bidentatae Radix" and "Os Draconis-Pheretima-Chuanxiong Rhizoma". Through factor reliability analysis， new drug combinations were derived， such as "Gastrodiae Rhizoma-Polygoni Multiflori Caulis-Eucommiae Cortex-Taxilli Herba-Leonuri Herba"， "Achyranthis Bidentatae Radix-Uncariae Ramulus cum Uncis"， "Scutellariae Radix-Glycyrrhizae Radix et Rhizoma-Margarita-Prunellae Spica"， "Os Draconis-Pheretima-Bombyx Batryticatus"， "Chrysanthemi Flos-Chuanxiong Rhizoma"， "Poria-Acori Tatarinowii Rhizoma"， and "Paeoniae Radix Alba-Gardeniae Fructus-Sclerotium Poriae Pararadicis". The Haliotidis Concha-Oystreae Concha drug pair was mainly used to treat diseases with liver Yang hyperactivity syndrome， with hypertension accounting for 40.00%， migraines for 30.00%， and dizziness for 15.00%. In the treatment of liver Yang hyperactivity syndrome， the main categories of compatible drugs were liver-pacifying and wind-extinguishing ones （19.86%）， blood-activating and stasis-resolving ones （12.13%）， and spirit-calming ones （10.08%）. High-frequency drugs in the prescriptions function to reduce blood pressure through multiple pathways， such as increasing nitric oxide （NO） levels， downregulating angiotensin Ⅱ （Ang Ⅱ）， and inhibiting angiotensin-converting enzyme （ACE）. ConclusionThrough comprehensive analysis of the results， the Haliotidis Concha-Oystreae Concha drug pair is commonly used for hypertension with liver Yang hyperactivity syndrome， and is often combined with deficiency-tonifying， liver-pacifying and wind-extinguishing， heat-clearing， and spirit-calming drugs， aiming to simultaneously extinguish wind， relieve spasms， and pacify the liver to subdue Yang， while also clearing heat to relax bowels， stabilizing the mind， and enhancing the liver-pacifying and Yang-subduing effects of this drug pair.