This study explored the effect of the Mycobacterium tuberculosis(Mtb)PE/PPE family protein PE_PGRS37 on the growth of Mycobacterium smegmatis(Ms)and macrophage autophagy during Mtb infection.The pe_pgrs37 gene was amplified from Mtb genome through PCR,and the recombinant vector pAIN-PE_PGRS37 was successfully constructed through homologous recombi-nation.pAIN-PE_PGRS37 and pAIN were integrated into Ms through electroshock to construct pAIN-PGRS37/Ms and pAIN/Ms re-combinant bacteria.Western blotting indicated that the PE_PGRS37 protein was correctly expressed in pAIN-PE_PGRS37/Ms.The re-combinant bacteria were inoculated in 7H9/7H10 medium,and their colony morphology and growth curves were observed.No signifi-cant difference in colony morphology was observed between pAIN-PE_PGRS37/Ms and pAIN/Ms.The growth rate significantly in-creased between 10 and 16 h,and a plateau was reached at 26 h.After infection of U937 cells with pAIN-PE_PGRS37/Ms and pAIN/Ms,macrophage autophagy flow was detected with western blotting and immunofluorescence.In the pAIN-PE_PGRS37/Ms-infected group,compared with the pAIN/Ms-infected group,macrophage LC3-II and p62 protein expression was significantly up-regulated(P<0.001)and inhibited autophagosome and lysosome fusion.The intracellular survival of the recombinant bacteria was detected through colony counting,and pAIN-PE_PGRS37/Ms showed significantly greater survival in macrophages at 12 h,24 h,and 48 h than pAIN/Ms(P<0.05).Our results suggested that PE_PGRS37 protein promotes Mycobacterium survival in macrophages by blocking macro-phage autophagy flow,thus inhibiting macrophage autophagy.

This study explored the effect of the Mycobacterium tuberculosis(Mtb)PE/PPE family protein PE_PGRS37 on the growth of Mycobacterium smegmatis(Ms)and macrophage autophagy during Mtb infection.The pe_pgrs37 gene was amplified from Mtb genome through PCR,and the recombinant vector pAIN-PE_PGRS37 was successfully constructed through homologous recombi-nation.pAIN-PE_PGRS37 and pAIN were integrated into Ms through electroshock to construct pAIN-PGRS37/Ms and pAIN/Ms re-combinant bacteria.Western blotting indicated that the PE_PGRS37 protein was correctly expressed in pAIN-PE_PGRS37/Ms.The re-combinant bacteria were inoculated in 7H9/7H10 medium,and their colony morphology and growth curves were observed.No signifi-cant difference in colony morphology was observed between pAIN-PE_PGRS37/Ms and pAIN/Ms.The growth rate significantly in-creased between 10 and 16 h,and a plateau was reached at 26 h.After infection of U937 cells with pAIN-PE_PGRS37/Ms and pAIN/Ms,macrophage autophagy flow was detected with western blotting and immunofluorescence.In the pAIN-PE_PGRS37/Ms-infected group,compared with the pAIN/Ms-infected group,macrophage LC3-II and p62 protein expression was significantly up-regulated(P<0.001)and inhibited autophagosome and lysosome fusion.The intracellular survival of the recombinant bacteria was detected through colony counting,and pAIN-PE_PGRS37/Ms showed significantly greater survival in macrophages at 12 h,24 h,and 48 h than pAIN/Ms(P<0.05).Our results suggested that PE_PGRS37 protein promotes Mycobacterium survival in macrophages by blocking macro-phage autophagy flow,thus inhibiting macrophage autophagy.

Chronic myelomonocytic leukemia(CMML)complicated with immune thrombocytopenia(ITP)is rare.This article reports the clinical data of a patient with CMML complicated with ITP treated with a combination of venetoclax,ripertamab(an anti-CD20 monoclonal antibody),and hetrombopag.The coexistence mechanism of CMML and ITP needs to be further clarified.Venetoclax combined with anti-CD20 monoclonal antibody and thrombopoietin receptor agonist may be an effective strategy for the treatment of this complication.

Objective:To explore the changes of dynamic cerebral autoregulation ability in pilots exposed to acute positive acceleration(+ Gz) by transcranial Doppler combined with beat-to-beat blood pressure.Methods:A total of 26 pilots enrolled in the + 8Gz manned centrifuge trial at the Air Force Medical Center, Air Force Medical University from June to October 2022 were prospectively included. Blood pressure and heart rate were monitored in the resting state before the trial and within 5 min after centrifugation. Transcranial Doppler combined with noninvasive continuous beat-to-beat blood pressure monitor were used to detect bilateral middle cerebral artery blood flow velocity and beat-to-beat pulse pressure respectively. The transfer function analysis was applied to derive the parameters of cerebral blood flow autoregulation in each frequency band from 0.02 to 0.50 Hz, and the phase, gain and coherence were calculated. The above parameters were compared between resting state and after acute + 8Gz positive acceleration exposure.Results:Compared with the resting state, in all of the 26 pilots after acute + 8Gz positive acceleration exposure, the systolic and diastolic blood pressure and heart rate increased significantly ( P<0.001), the phase significantly increased and the gain significantly decreased in the ultra-low frequency band (0.02-0.07 Hz) ( P<0.05); whereas there were no statistical differences of gain and phase in the low frequency band (0.07-0.20 Hz) and the high frequency band (0.20-0.50 Hz) (all P>0.05). Conclusions:Transcranial Doppler combined with beat-to-beat pulse pressure can be used for the assessment of changes in immediate dynamic cerebral autoregulation after acute + Gz exposure, and transfer function analysis of ultra-low frequency band parameters is suitable for this type of evaluation.

Objective:To compare the values of medical image technologies in evaluating the tansperineal laser ablation (TPLA) in canine prostate.Methods:TPLA (3 W/600 J and 3 W/1 200 J) were operated in the prostate of six adult male beagles guided by transrectal ultrasound (TRUS). TRUS, transrectal contrast-enhanced ultrasound (TR-CEUS) and multiparameter magnetic resonance imaging (mpMRI) were used to evaluate the ablation on the day of TPLA, one week and one month after TPLA. The animals were sacrificed for pathology to calculate the volume of the ablation. SPSS 22.0 software was used for statistical analysis.Results:TRUS could be used to guide and observe the puncture and ablation process during TPLA. TR-CEUS and contrast enhanced MRI showed good consistency in the volume of ablation ( P>0.05). One month after TPLA, the ablation volume were (1.69±0.51)ml vs (1.73±0.36)ml vs (1.52±0.41)ml (3 W/600 J) and (2.23±0.54)ml vs (2.34±0.29)ml vs (2.19±0.34)ml (3 W/1 200 J) measured by the two medical image technologies and pathology, with good consistency ( P>0.05). Conclusions:TRUS can be used to guide and observe the puncture and ablation process during TPLA. TR-CEUS and mpMRI can be used for postoperative evaluation and follow-up of TPLA. The former has advantages of real-time and low price, which can be promoted and applied in clinical practice.

Objective To explore the expression of miRNA-181a (miR-181a) in patients with multiple myeloma (MM) and its effect on biological features of MM cells. Methods CD138+cells of bone marrow from 25 MM patients and 10 patients with hematological non-malignancies were purified by using immunomagnetic separation, and the expression of miR-181a in CD138+cells and MM cell lines including RPMI 8226, H929 and U266 were detected by real-time quantitative PCR. The effects of down-regulation and up-regulation of miR-181a expression on the biological characteristics of MM cells were studied with miR-181a antagomir and agomir. Results Compared with patients with hematological non-malignant diseases, the expression of miR-181a in CD138+ cells was upregulated in MM patients. Compared with CD138+ cells in hematological non-malignancies, high expressions of miR-181a were observed in RPMI 8226 and U266 myeloma cell line, while low expressions of miR-181a were observed in H929 cells. Down-regulation of miR-181a with 100 nmol/L miR-181a antagomir could inhibit the proliferation of U266 cells at 24,48 and 72 h [(67.1 ± 3.3) %vs. (50.5 ± 4.1) %, (71.5 ± 3.6) % vs. (52.3 ± 2.2) %, (78.1 ± 5.4) % vs. (69.5 ± 4.3) %, P < 0.05 respectively], whereas up-regulation of miR-181a with 100 nmol/L miR-181a agomir could significantly promote the proliferation of H929 cells at 24 h and 48 h [(21.2 ± 2.4) %vs. (38.5 ± 3.6) %, ( 61.3 ± 5.4) %vs. (82.2 ±6.9)%, P<0.01 respectively]. Cell cycle analysis showed that miR-181a antagomir made U266 cell cycle arrest in the G0/G1 phase. Meanwhile, susceptibility test results indicated that the apoptosis of U266 cells induced by doxorubicin, paclitaxel and 5-fluorouracil was increased when the proliferation of miR-181a expression was down-regulated with miR-181a antagomir. In migration assay, the data showed that down-regulation of miR-181a with miR-181a antagomir could inhibit the migration of U266 cells, and the proportion of migrated cells in the experimental group (62 ± 10) %was lower than that in the control group (89 ± 12) %(P< 0.05), whereas up-regulation of miR-181a with miR-181a agomir could improve the migration of H929 cells, and the proportion of migrated cells in the experimental group (242 ± 9) % was higher than that in the control group (98 ± 8)%(P<0.01). Conclusions The high expression of miR-181a expressed highly by MM cells may promote the proliferation, migration and drug resistance of myeloma cells, indicating that miR-181a could be an important prognostic biomarker candidate, and the application of gene silencing may improve the prognosis of MM.

Objective: To investigate the pathogen spectrum distribution and drug resistance of febrile neutropenic patients with hematological diseases in Shanghai. Methods: A retrospective study was conducted on the clinical isolates from the febrile neutropenic patients hospitalized in the departments of hematology in 12 general hospitals in Shanghai from January 2012 to December 2014. The drug susceptibility test was carried out by Kirby-Bauer method. WHONET 5.6 software was used to analyze pathogenic bacteria and drug susceptibility data. Results: A total of 1 260 clinical isolates were collected from the febrile neutropenic patients. Gram-positive bacteria accounted for 33.3% and Gram-negative bacteria accounted for 66.7%. Klebsiella pneumoniae (12.5%) , Stenotrophomonas maltophilia (9.5%) , Escherichia coli (9.1%) , Pseudomonas aeruginosa (8.7%) , Acinetobacter baumannii (6.6%) , Staphylococcus aureus (5.6%) and Enterococcus faecium (5.0%) were ranked in the first 7 of all pathogens. In the respiratory tract secretions specimens, non-fermented strains accounted for 56.2%. Stenotrophomonas maltophilia accounted for 15.2%. Enterobacteriaceae and coagulase-negative Staphylococci accounted for 42.3% (104/246) and 32.6% (85/246) respectively in blood samples. Enterobacteriaceae and Enterococcus bacteria accounted for 39.4% (76/193) and 28.5% (55/193) respectively in pus specimens. The detection rates of methicillin resistant Staphylococcus aureus (MRSA) and methicillin resistant coagulase negative Staphylococci (MRCNS) were 54.3% and 82.5%, respectively. Staphylococcus bacterial strain was not found to be resistant to linezolid, vancomycin and teicoplanin. The detection rate of Enterococcus vancomycin-resistant strains was 8.9%. Enterococcus was not detected resistance to oxazolidinone strains. Enterobacteriaceae bacteria were highly sensitive to carbapenems. The resistance rate of Pseudomonas aeruginosa to imipenem and meropenem was 34.1% and 15.8%, respectively. Stenotrophomonas maltophilia was more sensitive to minocycline hydrochloride, levofloxacin and sulfamethoxazole. The resistance rate of Acinetobacter baumannii only to cefoperazone-sulbactam was less than 10.0%. The antibiotic resistance rate of Klebsiella pneumoniae, Stenotrophomonas maltophilia, Pseudomonas aeruginosa and Acinetobacter baumanii to most of common antibiotics was lower than that of the CHINET surveillance. Conclusions The pathogenic strain distribution in common infection sites of febrile neutropenic patients was characterized. Bacterial resistance surveillance was better than the CHINET nationwide large sample surveillance in China.

Objective To evaluate the bacteriostatic effect of recombinant human lactoferrin(rhLF) on Helicobacter(H .) py‐lori and its influence on CagA ,Ure and gastric mucosal IL‐8 .Methods The minimum inhibitory concentration(MIC)and the influ‐ence of different drug concentrations on the proliferation of H .pylori were detected .The effects of rhLF on the mRNA and protein expressions of CagA and Ure in H .pylori were detected by RT‐PCR and Western blot ,respectively .The animal study :Balb/c mice were adopted and assigned randomly into four groups ,including the standard triple+rhLF(group A) ,rhLF(group B) ,standard tri‐ple(group C) and normal saline(group D) .The histopathological HE staining was used to observe the gastric inflammation and ELISA was used to detect the IL‐8 level of gastric tissue in each group .Results MIC was 0 .5 mg/mL ,moreover rhLF inhibited the bacterial growth and proliferation with a concentration‐dependent manner .rhLF could reduce the expression of H .pylori major viru‐lence factor CagA ,mRNA and protein of Ure .Comparing the group A with the group B ,C and D ,the gastric mucosal inflammation score and the IL‐8 levels of gastric tissue homogenates had statistically significant differences(P<0 .05) .Conclusion rhLF inhibits the growth and proliferation of H .pylori ,moreover inhibit the expression of major virulence factor CagA in H .pylori ,mRNA and protein of Ure in different degrees ,weakens its pathogenicity ,meanwhile reduces the IL‐8 level in mice gastric mucosa ,and allevi‐ates H .pylori related gastric mucosal inflammatory response .

In producing transgenic livestock, selectable marker genes (SMGs) are usually used to screen transgenic cells from numerous normal cells. That results in SMGs integrating into the genome and transmitting to offspring. In fact, SMGs could dramatically affect gene regulation at integration sites and also make the safety evaluation of transgenic animals complicated. In order to determine the deletion time and methods in the process of producing transgenic goats, the feasibility of deleting SMGs was explored by Cre/LoxP before or after somatic cell cloning. In addition, we compared the efficiency of protein transduction with plasmids co-transduction. We could delete 43.9% SMGs after screening out the transgenic cell clones, but these cells could not be applied to somatic cells cloning because of serious aging after two gene modifications. The SMG-free cells suitable for nuclear transfer were accessible by using the cells of transgenic goats, but this approach was more time consuming. Finally, we found that the Cre plasmid could delete SMGs with an efficiency of 7.81%, but about 30% in SMG-free cells had sequences of Cre plasmid. Compared with Cre plasmid, the integration of new exogenous gene could be avoided by TAT-CRE protein transduction, and the deletion rate of TAT-CRE transduction was between 43.9 and 72.8%. Therefore, TAT-Cre transduction could be an effective method for deleting selectable marker genes.
Animals ; Animals, Genetically Modified ; genetics ; Cloning, Organism ; veterinary ; Gene Knockout Techniques ; Gene Targeting ; methods ; Genes, Reporter ; Genetic Engineering ; Genetic Vectors ; genetics ; Goats ; genetics ; Integrases ; chemistry ; metabolism ; Recombination, Genetic ; Transgenes ; genetics

In the application of therapeutic antibodies, large molecular weight of antibodies is always a problem that prevents them from penetrating into tissues or binding to antigenic determinants. To overcome this problem, we investigated the function of the heavy chain variable domain of a monoclonal anti-VEGF human IgM antibody derived from the Five-Feature Translocus Mice. We cloned the cDNA of the heavy chain variable domain, which was then inserted into pET28a vector and expressed in Escherichia coli. After purification and renaturation of the denatured recombinant protein, we obtained a 16 kDa antibody fragment, which is named as rhVVH. By immunoassaying its VEGF-binding capability in vitro, we proved that rhVVH retains this activity as the complete IgM. Importantly, rhVVH is shown to inhibit the HUVEC cell proliferation in a concentration-dependent manner. Our results indicate that the single heavy chain variable domain might inherit part of the biological function of the complete IgM antibody, which provided a valuable potential in further research on antibody miniaturisation.
Amino Acid Sequence ; Animals ; Base Sequence ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Humans ; Immunoglobulin Heavy Chains ; biosynthesis ; genetics ; Immunoglobulin Variable Region ; biosynthesis ; genetics ; Mice ; Molecular Sequence Data ; Recombinant Proteins ; biosynthesis ; genetics ; Single-Chain Antibodies ; biosynthesis ; genetics ; Vascular Endothelial Growth Factor A ; genetics ; metabolism