Background:Chronic neutrophilic leukemia (CNL) is a rare myeloproliferative neoplasm (MPN), since the first description of CNL in 1920, more than 150 cases have reported in the literature. The World Health Organization (WHO) recognizes CNL, as a MPN and, for the frst time, provides recognized criteria to permit the operational classifcation of this poorly defned disease. Until recently, the molecular pathogenesis of CNL was unknown and the diagnosis was based on morphological aspects, clinical criteria and exclusion of known genetic entities like the Philadelphia translocation indicative of CML, or JAK2 mutations indicative of MPNs. Recent discovery of highfrequency granulocyte-colony stimulating factor receptor (CSF3R) mutations in CNL identifes a new major diagnostic criterion, and lend more specificity to the WHO diagnostic criteria for CNL, which are variably applied in routine clinical practice. In 2013 Maxson et al., and Pardanani and colleagues identified granulocyte-colony stimulating factor 3 receptor (CSF3R) mutations in 8 of 9 (89%), and in 13 of 13 (100%) patients with CNL, respectively. CSF3R mutations fall into 2 classes: nonsense or frameshift mutations that lead to premature truncation of the cytoplasmic tail of the receptor (truncation mutations) and point mutations in the extracellular domain of CSF3R (membrane proximal mutations). These nonsense or frameshift mutations truncate the cytoplasmic tail of CSF3R, impair its internalization,and alter its interactions with proteins such as SHP-1/2 and SOCS family members. These structural and functional alterations are thought to perturb the capacity of CSF3R to regulate granulocyte differentiation and to increase granulocytic proliferative capacity. Thetwo types of CSF3R mutations may have differential susceptibility to classes of tyrosine kinase inhibitors,with CSF3R truncation mutations showing activation of SRC family–TNK2 kinase signaling and sensitivity to dasatinib, and CSF3Rmembrane proximal mutations strongly activate the JAK/signal transducer and activator of transcription pathway and are sensitive to JAK kinase inhibitors such as ruxolitinib.The most common CSF3R mutation in CNL is themembrane proximal mutation: T618I. On September 2012 we got a case (a 67-years-old Chinese man), which had fulflled the WHO diagnostic criteria for CNL with a novel mutation site of colony stimulation factor 3 receptor (CSF3R). In our case was identifed a membrane proximal mutations CSF3RT618I and also a unreported novel mutation site of CSF3R-H54A in the CD34+ and CD15+ cell fractionsby sorting bone marrow samples (BD FACSAria™ III; BD Biosciences) using a PCR-based DNA pyrosequencing method. Thus, we sought to determine CSF3R-FL, CSF3R-T618I, CSF3R-H54A mutations have some correlation with molecular pathogenesis of CNL.Objective:To determine CSF3R-FL, CSF3R-T618I, CSF3RH54A mutations that have some signifcance on the molecular pathogenesis of CNL.Materials/Methods:1. Plasmid construction. Plasmids were constructed by PCR amplifcation of the insert, restriction digestion and ligation using standard molecular biology methods, briefly: the host vectors pLV-EF1α-EYFP-N, pLP-2, pVSV-G, pLP-1 gag pol were purifed with Omegabiotek maxi prep kit and digested by restriction enzymes (ECO RI, NOTI). The linearized vectors were purified from agarose gel using a AxyPrep TM DNA Gel Extraction Kit and the concentration of the samples was estimated on an agarose gel stained with ethidium bromide.The inserts (CSF3R-FL, CSF3R-T618I, CSF3R- H54A) were generated by PCR, the sequences of the primer-pairs used and the conditions of the PCR reactions. The amplifed DNA fragments were purifed from agarose gels using AxyPrep TM DNA Gel Extraction kits and digested by ECO RI, NOTI was used to linearize the acceptor vector. Enzymatic reactions in the case of the i) insert: 100–3000 ng purifed PCR product was digested by appropriate amount of enzyme and 4 µl of 10x reaction buffer in 40 µl of fnal volume for overnight at the appropriate temperature; ii) vector: 2000–4000 ng plasmid DNA was digested by appropriate amount of enzyme and 2 µl of 10x reaction buffer in 20 µl of fnal volume for 4 hours at the appropriate temperature. When necessary Research on the Influence of new oncogenic CSF3R mutations in Chronic Neutrophilic Leukemia Otgonbat Altangerel1, Ming Feng Zhao2, Wu Ri Mao21Department of Internal Medicine, Division of Hematology, Mongolian National University of Medical Sciences, Mongolia 2Department of Hematology, Tianjin First Central Hospital, First Central Clinical College of Tianjin Medical University,P.R. China11 the digested fragments were purifed again and the concentrations of the inserts were estimated on agarose gels, as described above. A 1:3 vector: insert molar ratio was used for the ligation reactions. Chemically competent DH5α Escherichia coli bacteria were transformed with the products of the ligation reactions and were grown on Luria Bertrani (LB) agar plates containing the required antibiotic, such as ampicillin (Sigma). A day later single colonies were picked from the plate, inoculated into and grown overnight in LB medium containing with ampicillin. Plasmids were purifed from the overnight cultures as above and tested by restriction mapping for the presence of the insert. Selected clones were sequenced by Sanger sequencing.2. Lentiviral packaging system we used 3 main components, such as the lentiviral expression vector(Plasmid DNA of CSF3R-FL, CSF3R-T618I), the lentiviral packaging plasmids (pLP-1, pLP-2 plasmids that encode for gag, pol, and rev from the HIV or FIV genome and pVSV-G), 293TNN producer cells. We seed 1X105 293TNN cells per 10 cm2 culture plate in 2-3 ml of culture medium containing DMEM medium supplemented with 4 mM L-glutamine, 4.5 g/l glucose, and fetal bovine serum (10%) without antibiotics. Grow for 18-24 hours at 37 °C with 5% CO 2 so that the cell density reaches ~60 - 80% confluency at the time of transfection. We used a GFP as a positive control, to confrm transfection experiment was successful. Then we collected the cell culture supernatant, which is containing infectious pseudoviral particles.3. Transduction of Pseudotyped Viral Particles into the primary cell of mouse. In the fnal step we have used the Mouse Colony Forming Unit Assay using MethoCultTM to assess the effects of CNL-associated oncogenes on the morphology and number of primary murine cells derived bone marrow. For this purpose cells are transduced with either control, which is without viral construct or a construct expressing the oncogene of interest (CSF3R-FL, CSF3R-T618I).Results:1. On the Plasmid construction step we successfully extracted and purifcated of recombinant plasmids ofCSF3R-FL and CSF3R-T618I cloning, but we still didexperiment to obtain the recombinant plasmid CSF3R- H54A cloning.2. After 24 hours of transfection 293TNN Cells with Packaging Plasmids and the Expression Construct, cells we visualized with green fluorescence protein under the fluorescence microscope.3. The both two of CSF3Rcloning were capable of transforming murine colony forming cells. After transforming, CFU-GM colonies were counted manually by light microscopy seven days after plating. We found that the membrane proximal mutation (T618I) transformed CFU-GM colonies number was more than the full length non-mutants (CSF3RFL), which indicates that T618 mutation of CSF3R conferred the clonal advantage of CNL leukemia cells.Conclusions:1. The establishment of the plasmid reconstruction, lentiviral packaging system and Mouse Colony Forming Unit Assay were done successfully.2. We confrmed the transformation capacity of the CSF3R mutations, especially CSF3R-T618I was higher than CSF3R-FL. This result demonstrates that T618 mutation of CSF3R conferred the clonal advantage of CNL leukemia cells.3. Further studies will be continued and are needed to prove the effects of the novel mutation site CSF3RH54A on the transduced murine bone marrow progenitor cells by using CFC assay

Objective To establish a convenient,accurate and practical method for detection of adefovir dipivoxil resistance-as- sociated mutation in hepatitis B virus:rtA181V/T/S and rtN236T mutations.Methods According to HBV complete sequences in GenBank,two pairs of primers were designed to amplify the region of HBV reverse transcriptase in order to introduce a BglI restriction site upon PCR product of wild type (wt) and a BseDI restriction site upon PCR product of rt236 mutant type.After amplification,the PCR products were digested with BglI and BseDI separately.We used this method to detect wild,rt181 mu- tant,rt236 mutant plasmids and 3 chronic hepatitis B patients' serum with obvious ADV resistance-associated mutations.We also tested the sensitivity of this method by mixing the wild and mutant plasmids in different proportions.Results The method could detect rt181 and rt236 mutations simultaneously.The result of RFLP analysis was in accordance with that of DNA se- quencing and cloning analysis.This method could detect the mutants even when they comprised only 10% of the total virus population.Conclusions The PCR-RFLP method with high sensitivity can detect rt181 and rt236 mutations simultaneously.It can be used for early detection of ADV resistance-associated mutation in hepatitis B virus.

OBJECTIVETo observe the effects of series of Muskone (the muskone includes Slender Dutchmanspipe Root, Inula Root and neither kind of Common Aucklandia Root) on the heart hemodynamics and myocardial consumption of oxygen in experimental dogs, and to explain its pharmacological action on cardiovascular system.

METHODArterial blood pressure, coronary blood flow, resistance in coronary artery, total peripheral resistance, work of left artrium and oxygen consumption index of the cardiac muscles were observed in anaesthetic dogs.

RESULTThe series of Muskone decreased arterial blood pressure significantly, dilated coronary artery and peripheral arteries significantly, increased coronary blood flow, decreased resistance in coronary artery, improved the work of left artrium, the oxygen availability of cardiac muscles and the complaisance of arteries in cardiac muscles.

Animals ; Aristolochia ; chemistry ; Asteraceae ; chemistry ; Blood Pressure ; drug effects ; Coronary Circulation ; drug effects ; Cycloparaffins ; isolation & purification ; pharmacology ; Dogs ; Drug Combinations ; Female ; Heart ; drug effects ; physiology ; Hemodynamics ; drug effects ; Inula ; chemistry ; Male ; Myocardium ; metabolism ; Oxygen Consumption ; drug effects ; Plant Roots ; chemistry ; Plants, Medicinal ; chemistry ; Vascular Resistance ; drug effects

OBJECTIVETo study the therapeutic effects of the series of Muskone (the Muskone includes Slender Dutchmanspipe Root, Tumuxiang, and not Slender Dutchmanspipe Root) on experimental myocardial infarct and pain in rats.

METHODCoronary artery ligation was applied for the model of myocardial infarct. Therapeutic effects were evaluated by measuring parameters of histomorphometry, blood plasm of ET, 6- keto-PGF1alpha and TXB2. Intraperitoneal injection acetic was applied for the model of ache, the frequency and eclipse period of writhing were evaluated its effect of resisting pain.

RESULTThe Muskone including Radix Aristolociae, the Muskone including Radix Inulae and the Muskone without Radix Aucklandiae all can decrease the area of myocardial infarction in rats, the level of TXB2, ET, and the frequency of writhing significantly. Also it can increase the level 6-keto-PGF1alpha, the ratio of 6-keto-PGF1alpha and TXB2. Single Radix Aristolociae or Radix Inulae only relieved pain.

CONCLUSIONThe Muskone including Radix Aristolociae, the Muskone including Radix Inulae and the Muskone without Radix Aucklandiae all have significant therapeutic effect on both myocardial infarction and pain, while single Radix Aristolociae or Radix Inulae only can relieve pain.

6-Ketoprostaglandin F1 alpha ; blood ; Animals ; Aristolochia ; chemistry ; Cycloparaffins ; isolation & purification ; pharmacology ; Drug Combinations ; Drugs, Chinese Herbal ; isolation & purification ; pharmacology ; Endothelins ; blood ; Female ; Inula ; chemistry ; Male ; Mice ; Mice, Inbred ICR ; Myocardial Infarction ; drug therapy ; Pain ; physiopathology ; Plants, Medicinal ; chemistry ; Rats ; Rats, Wistar ; Thromboxane B2 ; blood

OBJECTIVETo study the resistant rate of hepatitis B virus (HBV) to ADV and the dynamic evolution of HBV in lamivudine (Lam)-resistant chronic hepatitis B (CHB) patients.

METHODSTwenty-three Lam-resistant CHB patients were assigned to a 10mg/d ADV monotherapy for 68-116 weeks. The baseline and different time point blood samples after ADV monotherapy were analyzed for ADV-resistant mutations using direct sequencing of PCR products; the evolution of HBV mutations was examined by clonal analysis of serial samples from one patient infected with ADV-associated resistant HBV strains.

RESULTSThe cumulative incidence of genotypic ADV resistance at weeks 48 and 96 was 4.3% and 10.5% respectively respectively. The evolution analysis of HBV mutant strains in an ADV-resistant CHB patient showed that the proportion of YMDD mutants gradually decreased with rtA181S mutants increasing over time after ADV monotherapy, and that rtA181S+N236T mutants became the predominant strains during prolonged ADV monotherapy. The addition of Lam to the ongoing ADV treatment had poorer antiviral response in the patient with rtA181S or rtA181S+N236T mutant infection; one clone with multi-drug resistant mutations was selected during Lam and ADV combination therapy.

CONCLUSIONIncreased risk of adefovir resistance and selection of multi-drug resistant mutations are associated with long-term ADV monotherapy in patients with Lam-resistant chronic hepatitis B.

Adenine ; analogs & derivatives ; therapeutic use ; Adult ; Antiviral Agents ; therapeutic use ; Drug Resistance, Viral ; Evolution, Molecular ; Female ; Hepatitis B virus ; classification ; drug effects ; genetics ; Hepatitis B, Chronic ; drug therapy ; virology ; Humans ; Lamivudine ; pharmacology ; Male ; Middle Aged ; Organophosphonates ; therapeutic use

OBJECTIVETo observe the effects of the series of Muskone (the Muskone includes Slender Dutchmanspipe Root, Tumuxiang, and not Slender Dutchmanspipe Root) on myocardial ischemia, myocardial infarction and hematological index in experimental canines, and to explain the pharmacological action and characteristic of its therapeutic effect on ischemic heart disease.

METHODThe range and degree of myocardial ischemia was evaluated by epicardial electrogram mapping, and the range extent of myocardial infarction was determined by quantitate histology (N-BT staining method). Meanwhile, the changes of ET, TXB2, 6-Keto-PGF1alpha were determined to study the effects of the series of Muskone on myocardial ischemia, myocardial infarction and hematological index in experimental canines.

RESULTThe series of Muskone can improve myocardial ischemia and infarction in experimental canines, and relieve significantly the degree of myocardial ischemia (Sigma-ST) determined by epicardial electrogram mapping, decrease the range of myocardial ischemia (N-ST) determined by epicardial electrogram mapping and decrease infarction zone determined by N-BT staining method. And it has a significant inhibition on activity of ET induced by myocardial ischemia and infarction, and increases 6-Keto-PGF1alpha and 6-Keto-PGF1alpha/TXB2 induced by myocardial ischemia.

CONCLUSIONThe series of Muskone has significant therapeutic effect on myocardial infarction.

6-Ketoprostaglandin F1 alpha ; blood ; Animals ; Aristolochia ; chemistry ; Cycloparaffins ; isolation & purification ; pharmacology ; Dogs ; Drug Combinations ; Drugs, Chinese Herbal ; isolation & purification ; pharmacology ; Endothelins ; blood ; Female ; Inula ; chemistry ; Male ; Myocardial Infarction ; blood ; drug therapy ; pathology ; Myocardium ; pathology ; Plants, Medicinal ; chemistry ; Thromboxane B2 ; blood

OBJECTIVETo preliminarily explore the effect of combination of volar buttress plate with external fixator for the distal radial fractures of type C3 caused by high-energy injuries.

METHODSFrom January 2001 to June 2007, 13 patients with distal radial fracture of type C3, 9 males and 4 females aged from 26 to 47 (average 37 years), were treated with volar buttress plate combined with external fixator plus the techniques of K-wires and bone grafting as necessary, whose effects were evaluated preliminarily through comparing the volar tilt, radial inclination, radial shortening and wrist function.

RESULTSFollowed up from 7 to 29 months (average 18 months), the volar tilt, radial inclination, radial shortening and wrist function of all patients recovered remarkably. Nine patients achieved excellent and 4 good according to Sarmiento score (modified by Stewart) in the radiological manifestation, while 5 patients displayed excellent, 6 good, and 2 fair according to Gartland-Werley functional assessment system.

CONCLUSION1) Volar buttress plate could support the valor cortex in order to prevent comminuted fragment from displacing and maintain volar tilt and to provide the volar fulcrum for external fixator. 2) External fixator, with the assistance of volar fulcrum, could maintain the volar tilt and the height of distal radius and help unload the fossa. 3) Supplemental K-wires fixation and the bone graft may assist fracture stable.

Adult ; Bone Plates ; External Fixators ; Female ; Fracture Fixation ; Humans ; Male ; Middle Aged ; Radius ; injuries ; surgery ; Radius Fractures ; surgery

OBJECTIVES: Chronic suppurative otitis media (CSOM) and middle ear cholesteatoma (MEC) are the 2 most common chronic middle ear diseases. In the process of diagnosis and treatment, the 2 diseases are prone to misdiagnosis and missed diagnosis due to their similar clinical manifestations. High resolution computed tomography (HRCT) can clearly display the fine anatomical structure of the temporal bone, accurately reflect the middle ear lesions and the extent of the lesions, and has advantages in the differential diagnosis of chronic middle ear diseases. This study aims to develop a deep learning model for automatic information extraction and classification diagnosis of chronic middle ear diseases based on temporal bone HRCT image data to improve the classification and diagnosis efficiency of chronic middle ear diseases in clinical practice and reduce the occurrence of missed diagnosis and misdiagnosis. METHODS: The clinical records and temporal bone HRCT imaging data for patients with chronic middle ear diseases hospitalized in the Department of Otorhinolaryngology, Xiangya Hospital from January 2018 to October 2020 were retrospectively collected. The patient's medical records were independently reviewed by 2 experienced otorhinolaryngologist and the final diagnosis was reached a consensus. A total of 499 patients (998 ears) were enrolled in this study. The 998 ears were divided into 3 groups: an MEC group (108 ears), a CSOM group (622 ears), and a normal group (268 ears). The Gaussian noise with different variances was used to amplify the samples of the dataset to offset the imbalance in the number of samples between groups. The sample size of the amplified experimental dataset was 1 806 ears. In the study, 75% (1 355) samples were randomly selected for training, 10% (180) samples for validation, and the remaining 15% (271) samples for testing and evaluating the model performance. The overall design for the model was a serial structure, and the deep learning model with 3 different functions was set up. The first model was the regional recommendation network algorithm, which searched the middle ear image from the whole HRCT image, and then cut and saved the image. The second model was image contrast convolutional neural network (CNN) based on twin network structure, which searched the images matching the key layers of HRCT images from the cut images, and constructed 3D data blocks. The third model was based on 3D-CNN operation, which was used for the final classification and diagnosis of the 3D data block construction, and gave the final prediction probability. RESULTS: The special level search network based on twin network structure showed an average AUC of 0.939 on 10 special levels. The overall accuracy of the classification network based on 3D-CNN was 96.5%, the overall recall rate was 96.4%, and the average AUC under the 3 classifications was 0.983. The recall rates of CSOM cases and MEC cases were 93.7% and 97.4%, respectively. In the subsequent comparison experiments, the average accuracy of some classical CNN was 79.3%, and the average recall rate was 87.6%. The precision rate and the recall rate of the deep learning network constructed in this study were about 17.2% and 8.8% higher than those of the common CNN. CONCLUSIONS The deep learning network model proposed in this study can automatically extract 3D data blocks containing middle ear features from the HRCT image data of patients' temporal bone, which can reduce the overall size of the data while preserve the relationship between corresponding images, and further use 3D-CNN for classification and diagnosis of CSOM and MEC. The design of this model is well fitting to the continuous characteristics of HRCT data, and the experimental results show high precision and adaptability, which is better than the current common CNN methods.
Algorithms ; Ear Diseases ; Humans ; Neural Networks, Computer ; Retrospective Studies ; Tomography, X-Ray Computed/methods*

Nocathiacin I, a glycosylated thiopeptide antibiotic, displays excellent antibacterial activities against multidrug resistant bacterial pathogens. Previously, a novel nocathiacin I formulation for intravenous administration has been successfully developed and its aqueous solubility is greatly enhanced for clinical application. The purpose of the present study was to increase the fermentation titer of nocathiacin I and reduce or eliminate analogous impurities by screening the medium ingredients using response surface methodology. After a sysmatic optimization, a water-soluble medium containing quality-controllable components was developed and validated, resulting in an increase in the production of nocathiacin I from 150 to 405.8 mg·L at 150-L scale. Meanwhile, the analogous impurities existed in reported processes were greatly reduced or eliminated. Using optimized medium for fermentation, nocathiacin I with pharmaceutically acceptable quality was easily obtained with a recovery of 67%. In conclusion, the results from the present study offer a practical and efficient fermentation process for the production of nocathiacin I as a therapeutic agent.
Actinobacteria ; growth & development ; metabolism ; Anti-Bacterial Agents ; biosynthesis ; chemistry ; Bioreactors ; Culture Media ; Fermentation ; Intercellular Signaling Peptides and Proteins ; Peptides ; chemistry ; metabolism ; Quality Improvement